Anti-transferrin receptor antibodies and methods of use

ABSTRACT

The present invention relates to anti-transferrin receptor antibodies and methods of their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry pursuant to 35 U.S.C. § 371of International Application No. PCT/US2015/061405, filed Nov. 18, 2015,which claims the benefit of priority of U.S. Provisional Application No.62/081,827, filed Nov. 19, 2014, which is incorporated by referenceherein in its entirety for any purpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2015-11-18_01146-0042-00PCT_ST25.txt” created on Nov. 18, 2015, whichis 130,420 bytes in size. The information in the electronic format ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to anti-transferrin receptor antibodiesand methods of using the same.

BACKGROUND

Brain penetration of large molecule drugs is severely limited by thelargely impermeable blood-brain barrier (BBB). Among the many strategiesto overcome this obstacle is to utilize transcytosis traffickingpathways of endogenous receptors expressed at the brain capillaryendothelium. Recombinant proteins such as monoclonal antibodies havebeen designed against these receptors to enable receptor-mediateddelivery of large molecules to the brain. Strategies to maximize brainuptake while minimizing reverse transcytosis back to the blood, and toalso maximize the extent of accumulation after therapeutic dosing havebeen addressed with the finding that antibodies with low affinity to BBBreceptors offer the potential to substantially increase BBB transportand CNS retention of associated therapeutic moieties/molecules relativeto typical high-affinity antibodies to such receptors (Atwal et al.,Sci. Transl. Med. 3, 84ra43 (2011); Yu et al., Sci. Transl. Med. 25 May2011: Vol. 3, Issue 84, p. 84ra44). However, those antibodies did notspecifically bind to human and primate TfR.

SUMMARY

Monoclonal antibodies have vast therapeutic potential for treatment ofneurological or central nervous system (CNS) diseases, but their passageinto the brain is restricted by the blood-brain barrier (BBB). Paststudies have shown that a very small percentage (approximately 0.1%) ofan IgG circulating in the bloodstream crosses through the BBB into theCNS (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)), where the CNSconcentration of the antibody may be insufficient to permit a robusteffect. It was previously found that the percentage of the antibody thatdistributes into the CNS could be improved by exploiting BBB receptors(ie, transferrin receptor, insulin receptorand the like) (see, e.g.,WO9502421). For example, the anti-BBB receptor antibody can be mademultispecific to target one or more desired antigens in the CNS, or oneor more heterologous molecules can be coupled to the anti-BBB receptorantibody; in either case, the anti-BBB receptor antibody can assist indelivering a therapeutic molecule into the CNS across the BBB.

However, targeting a BBB receptor with a traditional specifichigh-affinity antibody generally resulted in limited increase in BBBtransport. It was later found by Applicants that the magnitude ofantibody uptake into and distribution in the CNS is inversely related toits binding affinity for the BBB receptor amongst the anti-BBBantibodies studied. For example, a low-affinity antibody to transferrinreceptor (TfR) dosed at therapeutic dose levels greatly improves BBBtransport and CNS retention of the anti-TfR antibody relative to ahigher-affinity anti-TfR antibody, and makes it possible to more readilyattain therapeutic concentrations in the CNS (Atwal et al., Sci. Transl.Med. 3, 84ra43 (2011)). Proof of such BBB transport was achieved using abispecific antibody that binds both TfR and the amyloid precursorprotein (APP) cleavage enzyme, β-secretase (BACE1). A single systemicdose of the bispecific anti-TfR/BACE1 antibody engineered using themethodology of the invention not only resulted in significant antibodyuptake in brain, but also dramatically reduced levels of brain Aβ₁₋₄₀compared to monospecific anti-BACE1 alone, suggesting that BBBpenetrance affects the potency of anti-BACE1. (Atwal et al., Sci.Transl. Med. 3, 84ra43 (2011); Yu et al., Sci. Transl. Med. 3, 84ra44(2011)).

Those data and experiments highlighted several causative mechanismsbehind increasing uptake of an antibody into the CNS using alower-affinity antibody approach. First, high affinity anti-BBB receptor(BBB-R) antibodies (e.g., anti-TfR^(A) from Atwal et al. and Yu et al.,supra) limit brain uptake by quickly saturating the BBB-R in the brainvasculature, thus reducing the total amount of antibody taken up intothe brain and also restricting its distribution to the vasculature.Strikingly, lowering affinity for the BBB-R improves brain uptake anddistribution, with a robust shift observed in localization from thevasculature to neurons and associated neuropil distributed within theCNS. Second, the lower affinity of the antibody for the BBB-R isproposed to impair the ability of the antibody to return to the vascularside of the BBB via the BBB-R from the CNS side of the membrane becausethe overall affinity of the antibody for the BBB-R is low and the localconcentration of the antibody on the CNS side of the BBB isnon-saturating due to the rapid dispersal of the antibody into the CNScompartment. Third, in vivo, and as observed for the TfR system,antibodies with less affinity for the BBB-R are not cleared from thesystem as efficiently as those with greater affinity for the BBB-R, andthus remain at higher circulating concentrations than theirhigher-affinity counterparts. This is advantageous because thecirculating antibody levels of the lower-affinity antibody are sustainedat therapeutic levels for a longer period of time than thehigher-affinity antibody, which consequently improves uptake of antibodyin brain for a longer period of time. Furthermore, this improvement inboth plasma and brain exposure may reduce the frequency of dosing in theclinic, which would have potential benefit not only for patientcompliance and convenience but also in lessening any potential sideeffects or off-target effects of the antibody and/or of a therapeuticcompound coupled thereto.

The low-affinity BBB-R antibodies described in the above-referenced workwere selected/engineered to avoid interference with the natural bindingbetween transferrin and the TfR, and thus to avoid potential irontransport-related side effects. Nonetheless, upon administration ofcertain of these antibodies in mice, some marked side effects wereobserved. The mice displayed a primary response of robust depletion ofreticulocyte populations accompanied by rapid onset acute clinicalsymptoms. Though the mice recovered from both the acute clinicalsymptoms and the decreased reticulocyte levels in due course, avoidingor otherwise mitigating this impact on reticulocytes is clearlydesirable for an anti-TfR antibody to be able to be used safely as atherapeutic molecule. It was found that the primary response to anti-TfRadministration (robust reticulocyte depletion and acute clinical signs)is driven in large part by the antibody-dependent cell-mediatedcytotoxicity (ADCC) activity of the antibody, while the residualreticulocyte depletion effect is mediated by the complement pathway.

These prior studies utilized mouse antibodies which bound specificallyto mouse TfR, but which did not specifically recognize primate or humanTfR. Accordingly, the invention provides antibodies and functional partsthereof which do specifically recognize both primate and human TfR, inorder to facilitate safety and efficacy studies in primates with theantibodies prior to therapeutic or diagnostic use in humans. In vitrostudies using a human erythroblast cell line and primary bone marrowcells treated with the anti-human TfR antibodies of the inventiondemonstrated that a robust depletion of TfR-positive erythroid cells isalso observable in human/primate cellular systems as it is in mice (see,e.g., Example 4). Accordingly, also provided herein are modifications tothe antibodies of the invention to greatly reduce or eliminate theunwanted reduction in the TfR-expressing reticulocyte population uponanti-TfR administration while still enabling the enhanced BBB transport,increased CNS distribution and CNS retention provided by theanti-human/primate TfR antibodies administered at therapeuticconcentrations. Several general approaches to mitigate the observedeffect of the anti-TfR antibodies of the invention on both the primaryand residual reticulocyte depletion are provided herein, and may be usedsingly or in combination.

In one approach, the effector function of the anti-human/cyno TfRantibody is reduced or eliminated in order to reduce or eliminate ADCCactivity. In another approach, the affinity of the anti-human/cyno TfRantibody for human or primate TfR is further lessened such thatinteractions of the antibody with the reticulocyte population are lessdetrimental to that population. A third approach is directed to reducingthe amount of anti-human/cyno TfR antibody that is present in the plasmato reduce exposure of the reticulocyte population to potentiallydetrimental concentrations of the antibody. A fourth approach seeks toprotect, stabilize and/or replenish reticulocyte populations such thatany potential depletion of the reticulocyte population in circulation orin bone marrow by administration of the anti-human/cyno TfR antibody isavoided, lessened, or mitigated.

Effector function reduction or elimination, as described herein, may beaccomplished by: (i) reduction or elimination of wild-type mammalianglycosylation of the antibody, (for example, by producing the antibodyin an environment where such glycosylation cannot occur, by mutating oneor more carbohydrate attachment points such that the antibody cannot beglycosylated, or by chemically or enzymatically removing one or morecarbohydrates from the antibody after it has been glycosylated); (ii) byreduction or elimination of the Fc receptor-binding capability of theanti-human/cyno TfR antibody (for example, by mutation of the Fc region,by deletion within the Fc region or elimination of the Fc region); or(iii) by utilization of an antibody isotype known to have minimal or noeffector function (ie., including but not limited to IgG4).

Decreasing antibody complement activation, as described herein, may beaccomplished by reduction or elimination of the C1q binding capabilityof the anti-human/cyno TfR antibody (for example, by mutation of,deletion within or elimination of the Fc region, or by modifying thenon-Fc portion of the anti-human/cyno TfR antibody), or by otherwisesuppressing activation or activity of the complement system (forexample, by co-administering one or more complement pathway activationor complement pathway activity inhibitors).

When binding of anti-human/cyno TfR antibody to human or cyno TfR onreticulocytes or other cell types expressing high levels of TfR triggerstheir depletion, as with the anti-human/cyno TfR antibodies exemplifiedherein, reduction of binding of the antibodies to the human or cyno TfRon the reticulocytes or other cell types should in turn decrease theamount of reticulocyte or other cell type depletion in circulation or inbone marrow observed upon antibody administration. The affinity of theanti-human/cyno TfR antibody for primate or human TfR may be modifiedusing any of the methods described herein and as shown in the Examples.

Reducing the amount of anti-human/cyno TfR antibody present in theplasma in order to reduce exposure of the reticulocyte population topotentially detrimental concentrations of the antibody may beaccomplished in several ways. One method is to simply decrease theamount of the antibody that is dosed, potentially while also increasingthe frequency of the dosing, such that the maximal concentration in theplasma is lowered but a sufficient serum level is maintained forefficacy, while still below the threshold of the cell-depleting sideeffect. Another method, which may be combined with dosing modifications,is to select or engineer an anti-TfR antibody that has pH-sensitivebinding to TfR such that it binds to cell surface TfR in the plasma atpH 7.4 with desirably low affinity as described herein, but uponinternalization into an endosomal compartment, such binding to TfR israpidly and significantly reduced at the relatively lower pH of thatcompartment (pH 5.5-6.0). Such dissociation may protect the antibodyfrom antigen-mediated clearance, or increase the amount of antibody thatis either delivered to the CNS or recycled back across the BBB—in eithercase, the effective concentration of the antibody is increased relativeto an anti-TfR antibody that does not comprise such pH sensitivity,without increasing the administered dose of the antibody, and in turnpotentially permitting a lower dose of the antibody with concomitantlylesser risk of side effects.

Protecting, stabilizing and/or replenishing reticulocyte populations maybe accomplished using pharmaceutical or physical methods. In addition tothe anti-human/cyno TfR antibody, at least one further therapeutic agentmay be coadministered (simultaneously or sequentially) that mitigatesnegative side effects of the antibody on reticulocyte populations.Examples of such therapeutic agents include, but are not limited to,erythropoietin (EPO), iron supplements, vitamin C, folic acid, andvitamin B12. Physical replacement of red blood cells (ie, reticulocytes)is also possible by, for example, transfusion with similar cells, whichmay be from another individual of similar blood type or may have beenpreviously extracted from the subject to whom the anti-human/cyno TfRantibody is administered.

One of ordinary skill in the art will appreciate that any combination ofthe foregoing methods may be employed to engineer an antibody (and/ordosage regimen for same) with the optimum balance between (i) thedesirably low affinity for primate or human TfR that will maximizetransport of the antibody and any conjugated compounds into the CNS;(ii) the affinity of the conjugated compound (including as a nonlimitingexample, a second or further antigen-binding specificity in theanti-human/cyno TfR antibody) for its CNS antigen, since this isrelevant to the amount of the compound that needs to be present in theCNS to have a therapeutic effect; (iii) the clearance rate of theanti-human/cynoTfR antibody; (iv) the liability of theanti-TfR/conjugated compound at low pH to facilitate release of theconjugated compound on the CNS/brain side of the BBB, and (v) the impacton reticulocyte populations.

It will also be appreciated that the reticulocyte-depleting effectrecognized herein of anti-TfR antibody administration may be useful inthe treatment of any disease or disorder where overproliferation ofreticulocytes is problematic. For example, in congenital polycythemia orneoplastic polycythemia vera, raised red blood cell counts due tohyperproliferation of, e.g., reticulocytes, results in thickening ofblood and concomitant physiological symptoms. Administration of ananti-human/cyno TfR antibody of the invention wherein at least partialeffector function of the antibody was preserved would permit selectiveremoval of immature reticulocyte populations without impacting normaltransferrin transport into the CNS. Dosing of such an antibody could bemodulated such that acute clinical symptoms could be minimized (ie, bydosing at a very low dose or at widely-spaced intervals), aswell-understood in the art.

Anti-TfR/BACE1 and anti-TfR/Abeta are each promising and noveltherapeutic candidates for the treatment of Alzheimer's disease.Furthermore, receptor mediated transport (RMT)-based bispecifictargeting technology opens the door for a wide range of potentialtherapeutics for CNS diseases. The invention provides methods ofengineering BBB-penetrant therapeutics that greatly improve transportacross the BBB and CNS distribution of the therapeutic without depletionof reticulocytes.

Accordingly, in a first embodiment, the invention provides an isolatedantibody that binds to human transferrin receptor (TfR) and primate TfR,wherein the antibody does not inhibit the binding of transferrin to TfR.In some aspects, the binding is specific binding. In another aspect, theantibody further does not inhibit the binding of human hemachromatosisprotein (“HFE”) to TfR. In some aspects, the binding is specificbinding. In some aspects, the antibody is a monoclonal antibody. Inanother aspect, the antibody is a human antibody. In another aspect, theantibody is a humanized antibody. In another aspect, the antibody is achimeric antibody. In another aspect, the antibody is an antibodyfragment that binds human TfR and primate TfR. In another aspect, theprimate TfR is from cynomolgous monkey.

In any of the embodiments described herein, the antibody comprises aheavy chain variable region (VH) sequence of SEQ ID NO: 158 or 159. Inany of the embodiments described herein, the antibody further comprisesa light chain variable region (VL) sequence of SEQ ID NO: 162 or 163. Insome embodiments, the antibody comprises a heavy chain variable regionamino acid sequence of SEQ ID NO: 158 and a light chain variable regionamino acid sequence of SEQ ID NO: 162, or a heavy chain variable regionamino acid sequence of SEQ ID NO: 159 and a light chain variable regionamino acid sequence of SEQ ID NO: 163.

In some aspects of the above embodiment, the antibody is coupled to atherapeutic compound. In another aspect of the above embodiment, theantibody is coupled to an imaging agent or a label. In one such aspect,the antibody is a multispecific antibody and the therapeutic compoundoptionally forms one portion of the multispecific antibody. In one suchaspect, the multispecific antibody comprises a first antigen bindingsite which binds TfR and a second antigen binding site which binds abrain antigen. In one such aspect, the brain antigen is selected fromthe group consisting of: beta-secretase 1 (BACE1), Abeta, epidermalgrowth factor receptor (EGFR), human epidermal growth factor receptor 2(HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin,prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6),amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), andcaspase 6. In another such aspect, the multispecific antibody binds bothTfR and BACE1. In another such aspect, the multispecific antibody bindsboth TfR and Abeta. In another such aspect, the therapeutic compound isa neurological disorder drug.

In some embodiments, the multispecific antibody comprises a first heavychain comprising the sequence of SEQ ID NO: 160 and a first light chaincomprising the sequence of SEQ ID NO: 161.

In some aspects of the above embodiment, the invention provides anisolated nucleic acid encoding any of the foregoing antibodies. Inanother aspect, the invention provides a host cell comprising suchnucleic acid. In another aspect, the invention provides a method ofproducing any of the foregoing antibodies comprising culturing such hostcell so that the antibody is produced and optionally further comprisingrecovering the antibody from the host cell.

In some aspects of the above embodiment, the invention provides apharmaceutical formulation comprising any of the foregoing antibodiesand a pharmaceutically acceptable carrier.

In some aspects of the above embodiment, the invention provides any ofthe foregoing antibodies for use as a medicament. In another aspect ofthe above embodiment, the invention provides the use of any of theforegoing antibodies in the manufacture of a medicament for treating aneurological disorder. In one such aspect, the neurological disorder isselected from the group consisting of a neuropathy disorder, aneurodegenerative disease, cancer, an ocular disease disorder, a seizuredisorder, a lysosomal storage disease, amyloidosis, a viral or microbialdisease, ischemia, a behavioral disorder, and CNS inflammation.

In another aspect of the above embodiment, the invention provides any ofthe foregoing antibodies for use in treating a neurological disorder. Inone such aspect, the neurological disorder is selected from the groupconsisting of a neuropathy disorder, a neurodegenerative disease,cancer, an ocular disease disorder, a seizure disorder, a lysosomalstorage disease, amyloidosis, a viral or microbial disease, ischemia, abehavioral disorder, and CNS inflammation.

In another aspect of the above embodiment, the invention provides any ofthe foregoing antibodies for use in transporting one or more compoundsacross the BBB. In another aspect of the above embodiment, use of any ofthe foregoing antibodies in the manufacture of a medicament fortransporting one or more compounds across the BBB is provided.

In some aspects of the above embodiment, a method of transporting acompound across the BBB in a subject is provided, comprising exposingany of the foregoing antibodies to the BBB such that the antibodytransports the compound coupled thereto across the BBB. In another suchaspect, the BBB is in a human subject. In another such aspect, the doseamount and/or frequency of administration is modulated to reduce theconcentration of antibody to which red blood cells are exposed. Inanother such aspect, the method further comprises the step of monitoringthe subject for depletion of red blood cells. In another such aspect,the antibody coupled to the compound is administered at a therapeuticdose. In one such aspect, the therapeutic dose is TfR-saturating. Inanother such aspect, administration of the antibody is at a dose and/ordose frequency calibrated to minimize acute clinical symptoms of theantibody administration.

In another aspect of the above embodiment, a method of increasingexposure of the CNS of a subject to a compound is provided, comprisingexposing any of the foregoing antibodies to the BBB such that theantibody transports the compound coupled thereto across the BBB. Inanother such aspect, the BBB is in a human subject. In another suchaspect, the dose amount and/or frequency of administration is modulatedto reduce the concentration of antibody to which red blood cells areexposed. In another such aspect, the method further comprises the stepof monitoring the subject for depletion of red blood cells. In anothersuch aspect, the antibody coupled to the compound is administered at atherapeutic dose. In one such aspect, the therapeutic dose isTfR-saturating. In another such aspect, administration of the antibodyis at a dose and/or dose frequency calibrated to minimize acute clinicalsymptoms of the antibody administration.

In some aspects of the above embodiment, a method of increasingrentention in the CNS of a compound administered to a subject isprovided, comprising exposing any of the foregoing antibodies to the BBBsuch that the retention in the CNS of the compound is increased. Inanother such aspect, the BBB is in a human subject. In another suchaspect, the dose amount and/or frequency of administration is modulatedto reduce the concentration of antibody to which red blood cells areexposed. In another such aspect, the method further comprises the stepof monitoring the subject for depletion of red blood cells. In anothersuch aspect, the antibody coupled to the compound is administered at atherapeutic dose. In one such aspect, the therapeutic dose isTfR-saturating. In another such aspect, administration of the antibodyis at a dose and/or dose frequency calibrated to minimize acute clinicalsymptoms of the antibody administration.

In some aspects of the above embodiment, a method of treating aneurological disorder in a mammal is provided, comprising treating themammal with any of the foregoing antibodies. In one such aspect, theneurological disorder is selected from the group consisting of aneuropathy disorder, a neurodegenerative disease, cancer, an oculardisease disorder, a seizure disorder, a lysosomal storage disease,amyloidosis, a viral or microbial disease, ischemia, a behavioraldisorder, and CNS inflammation. In another such aspect, the neurologicaldisorder is in a human subject. In another such aspect, the dose amountand/or frequency of administration is modulated to reduce theconcentration of antibody to which the re blood cells are exposed. Inanother such aspect, the method further comprises the step of monitoringthe subject for depletion of red blood cells. In another such aspect,the antibody coupled to the compound is administered at a therapeuticdose. In one such aspect, the therapeutic dose is TfR-saturating. Inanother such aspect, administration of the antibody is at a dose and/ordose frequency calibrated to minimize acute clinical symptoms of theantibody administration.

In another embodiment, the invention provides an isolated antibody thatbinds to human TfR and primate TfR, wherein the antibody does notinhibit the binding of transferrin to TfR, and wherein one or moreproperties of the antibody have been modified to reduce or eliminate theimpact of the antibody on reticulocytes and/or reduce the severity orpresence of acute clinical symptoms in a subject or mammal treated withthe antibody. In some aspects, the binding is specific binding. Inanother aspect, the antibody further does not inhibit the binding of HFEto TfR. In some aspects, the antibody is a monoclonal antibody. Inanother aspect, the antibody is a human antibody. In another aspect, theantibody is a humanized antibody. In another aspect, the antibody is achimeric antibody. In another aspect, the antibody is an antibodyfragment that binds human TfR and primate TfR. In another aspect, theprimate TfR is from cynomolgous monkey.

In some aspects of the above embodiment, the one or more properties ofthe antibody are selected from the effector function of the antibody Fcregion, the complement activation function of the antibody and theaffinity of the antibody for TfR. In one such aspect, the property isthe effector function of the antibody Fc region. In another such aspect,the property is the complement activation function of the antibody. Inanother such aspect, the property is the affinity of the antibody forTfR. In one such aspect, the effector function or complement activationfunction has been reduced or eliminated relative to a wild-type antibodyof the same isotype. In some aspects, the effector function is reducedor eliminated by a method selected from reduction of glycosylation ofthe antibody, modification of the antibody isotype to an isotype thatnaturally has reduced or eliminated effector function, and modificationof the Fc region.

In one such aspect, the effector function is reduced or eliminated byreduction of glycosylation of the antibody. In one such aspect, theglycosylation of the antibody is reduced by a method selected from:production of the antibody in an environment that does not permitwild-type glycosylation; removal of carbohydrate groups already presenton the antibody; and modification of the antibody such that wild-typeglycosylation does not occur. In one such aspect, the glycosylation ofthe antibody is reduced by a production of the antibody in anenvironment that does not permit wild-type glycosylation, such asproduction in a non-mammalian cell production system or where theantibody is produced synthetically. In one such aspect, the antibody isproduced in a non-mammalian cell production system. In another suchaspect, the antibody is produced synthetically. In another such aspect,the glycosylation of the antibody is reduced by modification of theantibody such that wild-type glycosylation does not occur, such aswherein the Fc region of the antibody comprises a mutation at position297 such that the wild-type asparagine residue at that position isreplaced with another amino acid that interferes with glycosylation atthat position.

In another such aspect, the effector function is reduced or eliminatedby at least one modification of the Fc region. In one such aspect, theeffector function or complement activation function is reduced oreliminated by deletion of all or a portion of the Fc region, or byengineering the antibody such that it does not include an Fc region ornon-Fc region competent for effector function or complement activationfunction. In another such aspect, the at least one modification of theFc region is selected from: a point mutation of the Fc region to impairbinding to one or more Fc receptors selected from the followingpositions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278,289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329,333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435,437, 438, and 439; a point mutation of the Fc region to impair bindingto C1q selected from the following positions: 270, 322, 329, and 321;eliminating some or all of the Fc region, and a point mutation atposition 132 of the CH1 domain. In one such aspect, the modification isa point mutation of the Fc region to impair binding to C1q selected fromthe following positions: 270, 322, 329, and 321. In another such aspect,the modification is elimination of some or all of the Fc region. Inanother such aspect, complement-triggering triggering function isreduced or eliminated by deletion of all or a portion of the Fc region,or by engineering the antibody such that it does not include an Fcregion that engages the complement pathway. In one such aspect, theantibody is selected from a Fab or a single chain antibody. In anothersuch aspect, the non-Fc region of the antibody is modified to reduce oreliminate activation of the complement pathway by the antibody. In onesuch aspect, the modification is a point mutation of the CH1 region toimpair binding to C3. In one such aspect, the point mutation is atposition 132 (see, e.g., Vidarte et al., (2001) J. Biol. Chem. 276(41):38217-38223).

In some aspects, the antibody the half-life of the antibody is increasedby a modification in the FcRn binding region. In some aspects, themodification is a substitution in an amino acid selected from thefollowing positions: 251 256, 285, 290, 308, 314, 385, 389, 428, 434,436, 238, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,362, 376, 378, 380, 382, 413, 424 or 434. In some aspects themodification is a substitution selected from the following: M252Y,S254T, T256E, N434A and Y436I.

In some aspects, the antibody is combined with a further compound thatmitigates or contributes to the reduction of impact on reticulocytelevels or acute clinical symptoms. In one such aspect, the furthercompound protects reticulocytes from antibody-related depletion orsupports the growth, development, or reestablishment of reticulocytes.In another such aspect, the further compound is selected fromerythropoietin (EPO), an iron supplement, vitamin C, folic acid, andvitamin B12, or is red blood cells or reticulocytes.

In some aspects of the above embodiment, the affinity of the antibodyfor TfR is decreased, as measured relative to a wild-type antibody ofthe same isotype not having lowered affinity for TfR. In one suchaspect, the antibody has a KD or IC50 for TfR of about 1 pM to about 100μM, or about 10 nM to 100 nM, or about 20 nM to 100 nM. In anotheraspect, the dose amount and/or frequency of administration of theantibody is modulated to reduce the concentration of the antibody towhich the red blood cells are exposed.

In another embodiment, a method of decreasing clearance of a compoundadministered to a subject is provided, wherein the compound is coupledto an antibody which binds with low affinity to TfR, such that theclearance of the compound is decreased, and wherein reduction of redblood cell levels in the subject upon compound-coupled antibodyadministration to the subject is decreased or eliminated.

In another embodiment, a method of optimizing the pharmcokinetics and/orpharmacodynamics of a compound to be efficacious in the CNS in a subjectis provided, wherein the compound is coupled to an antibody which bindswith low affinity to TfR, and the antibody is selected such that itsaffinity for TfR after coupling to the compound results in an amount oftransport of the antibody conjugated to the compound across the BBB thatoptimizes the pharmacokinetics and/or pharmacodynamics of the compoundin the CNS, wherein reduction of red blood cell levels in the subjectupon compound-coupled antibody administration to the subject isdecreased or eliminated.

It will be understood that any of the foregoing methods and compositionsof the invention may be combined with one another and/or with thefurther aspects of the invention described in the specification herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a three-dimensional crystal structure of a TfR dimer incomplex with Tf, based on the pdb file 3SM9. The non-Tf-binding apicalregion of TfR is labeled.

FIGS. 2A-2B depict FACS analysis of mouse hybridoma parental clonesupernatants binding to human and cynomolgus TfR transiently expressedin 293 cells in the presence of 1 μM human holo-Tf. Unless otherwiseindicated, the filled grey trace in each graph is background from thedetection antibody, the medium grey trace is binding to 293 cells thatendogenously express basal levels of human TfR, the bold black tracerepresents binding to transiently expressed human TfR and the thin greytrace represents binding to transiently expressed cyno TfR.

FIG. 2C depicts the results of human/cynomolgous cross-reactive antibodycompetition assays as described in Example 1. Nine of the fourteenclones were found to block binding of the apical binding antibodydisplayed on phage.

FIGS. 3A-1, 3A-2, 3B-1, 3B-2, 3C-1, 3C-2, 3D-land 3D-2 depict the heavyand light chain variable region sequences of hybridoma clones that bindto apical and non-apical regions of TfR. The sequences can be furthersubdivided by epitope and sequence similarity into class I-III (apicalbinders) and class IV (non-apical binders). The HVRs according to Kabatare indicated by underlining.

FIGS. 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1 and 4D-2 depictalignments of humanized sequences for (A) 15G11, (B) 7A4/8A2, (C) 7G7and (D) 16F6. Each mouse light or heavy variable domain sequence (secondline) is aligned to the closest human germline or consensus variabledomain (first line). The humanized version for each antibody is shown atthe bottom (third line). Differences from the human germline orconsensus sequences are shaded. HVR sequences that were grafted into thehuman framework are boxed. CDR definitions according to Kabat areindicated.

FIGS. 4E-1 and 4E-2 show that for the class I-III groups of antibodies,variant forms of the antibodies with modifications at one or moreresidues of an FR retained affinity and binding specifity.

FIG. 5 depicts the binding of hu7A4.v15, hu15G11.v5 and hu7G7.v1 tohuTfR in the presence of 6.3 μM holo-Tf. Antibody binding to immobilizedhuTfR is shown in the presence (open symbols and dashed lines) orabsence (filled symbols and solid lines) of 6.3 μM holo-Tf.

FIGS. 6A-B depict the results of the HFE—HuTfR binding and the HFEblocking assays described in Example 1. FIG. 6A shows the binding ofantibody to increasing concentrations of huTfR captured via immobilizedHFE. FIG. 6B shows the binding of huTfR to immobilized HFE in thepresence of increasing concentrations of antibody.

FIG. 7A-B depict binding analyses of 15G11.v5 and 7A4.v5 IgG and Fab Alavariants on cyno and human TfR, demonstrating the effects on affinity ofAla mutations in CDR-L3 and CDR-H3 of each antibody assessed as IgG byELISA binding and IgG or Fab by SPR analysis to immobilized human orcyno TfR, as described in Example 2.

FIGS. 8A-B and FIGS. 9A-B depict the results of experiments assessingthe impact of effector function status on ADCC activity of anti-humanTfR (“anti-hTFR”) antibodies in primary human bone marrow mononuclearcells or in a human erythroblast cell line, as described in Example 4.

FIG. 10 depicts the dosing and sampling scheme for the primate studydescribed in Example 5.

FIGS. 11A-11B depict the pharmacokinetic results of the experimentsdescribed in Example 5, specifically individual and group meananti-TfR¹/BACE1, anti-TfR²/BACE1 and anti-gD serum concentrations versustime following a single IV bolus administration at 30 mg/kg incynomolgus monkeys in serum (FIG. 11A) and CSF (FIG. 11B).

FIGS. 12A-12E depict the pharmacodynamic results of the experimentsdescribed in Example 5, specifically individual and group meananti-TfR¹/BACE1, anti-TfR²/BACE1 and anti-gD plasma (A) or CSF (B-E)concentrations versus time following a single IV bolus administration at30 mg/kg in cynomolgus monkeys. The upper panels show Abetal-40 levelsin plasma (FIG. 12A) and CSF (FIG. 12B), while the lower panels showsoluble APPα levels (FIG. 12C), soluble APPβ levels (FIG. 12D), andsAPPβ/sAPPα ratio (FIG. 12E) over time.

FIGS. 13A-13D depict the results of hematological sampling performedduring the studies described in Example 5. At each of the indicated timepoints, total reticulocytes (FIG. 13A), red blood cells (FIG. 13B),hemoglobin (FIG. 13D) and the percentage of immature reticulocytes inthe total reticulocyte pool (FIG. 13C) were measured using standardtechniques.

FIG. 14 depicts the dosing and sampling scheme for the primate studydescribed in Example 6.

FIGS. 15A-15B depict the pharmacodynamic results (A) and brain antibodyconcentrations (B) of the experiments described in Example 6.Specifically, FIG. 15A shows individual and group mean anti-TfR¹/BACE1,anti-TfR²/BACE1, anti-gD, and anti-BACE1 ration of sAPPβ/sAPPα in CSFversus time following a single IV bolus administration at 30 mg/kg incynomolgus monkeys. FIG. 15B show individual anti-TfR¹/BACE1,anti-TfR²/BACE1, anti-gD, and anti-BACE1 concentrations of antibody invarious brain regions at 24 hours post-dose.

FIGS. 16A-B depict the light and heavy chain amino acid sequences ofanti-BACE1 clone YW412.8 obtained from a naïve sort of the naturaldiversity phage display library and affinity-matured forms of YW412.8.FIG. 16A depicts the variable light (VL) sequence alignments (SEQ IDNOs. 132-137). FIG. 16B depicts the variable heavy (VH) sequencealignments (SEQ ID Nos. 138-139). In both figures, the HVR sequences foreach clone are indicated by the boxed regions, with the first boxindicating HVR-L1 (FIG. 16A) or HVR-H1 (FIG. 16B), the second boxindicating HVR-L2 (FIG. 16A) or HVR-H2 (FIG. 16B), and the third boxindicating HVR-L3 (FIG. 16A) or HVR-H3 (FIG. 16B).

FIGS. 17A-B depict the light and heavy chain amino acid sequences ofanti-BACE1 antibody clone Fab 12 obtained from a naïve sort of asynthetic diversity phage display library and affinity-matured forms ofFab 12. FIG. 17A depicts the light chain sequence alignments (SEQ IDNOs. 140-143). FIG. 17B depicts the heavy chain sequence alignments (SEQID NO. 144). In both figures, the HVR sequences for each clone areindicated by the boxed regions, with the first box indicating HVR-L1(FIG. 17A) or HVR-H1 (FIG. 17B), the second box indicating HVR-L2 (FIG.17A) or HVR-H2 (FIG. 17B), and the third box indicating HVR-L3 (FIG.17A) or HVR-H3 (FIG. 17B).

FIGS. 18A-B depict the heavy chain (FIG. 18A; SEQ ID NO. 145) and lightchain (FIG. 18B; SEQ ID NO. 146) of an exemplary anti-Abeta antibody.

FIG. 19 depicts the pharmacokinetic properties of Anti-TfR¹/BACE1,Anti-Tfr^(52A)/BACE1 and Anti-TfR^(53A)/BACE1 as described in Example 5.

FIG. 20 depicts the pharmacokinetic properties of the murine IgG2aAnti-TfR^(D)/BACE1 and Anti-gD antibodies with the Fc effector functionLALAPG mutations as described in Example 7.

FIG. 21 depicts the total and immature reticulocyte count in mice 24hours after administration of a 50 mg/kg dose of the murine IgG2aAnti-TfR^(D)/BACE1 and Anti-gD antibodies with the Fc effector functionLALAPG mutations as described in Example 7.

FIG. 22 depicts the total reticulocyte count in mice 24 hours afteradministration of a 50 mg/kg dose of the anti-TfR^(52A)/BACE1 (N297G),anti-TfR^(52A)/BACE1 (LALAPG), anti-TfR⁵²A/BACE1 (LALAPG/YTE),TfR⁵²A/BACE1 (LALAPG/AI) antibodies in human transferrin receptorknock-in mice as described in Example 8.

FIG. 23 depicts the results of experiments assessing the impact ofeffector function status on ADCC activity of anti-TfR/gD, anti-TfR/BACE1(N297G), anti-TfR/BACE1 (LALAPG), anti-TfR-BACE1 (N297G/434A/436I) andanti-TfR/BACE1 (LALAPG/YTE) antibodies in primary human bone marrowmononuclear cells or in a human erythroblast cell line, as described inExample 8.

FIG. 24 depicts the heavy and light chain variable region sequences of1511Gv.5 (light chain—SEQ ID NO: 152 and heavy chain—SEQ ID NO: 108) andaffinity variants 15G11.52A (light chain—SEQ ID NO:152 and heavychain—SEQ ID NO: 153), 15G11.53A (light chain—SEQ ID NO: 152 and heavychain—SEQ ID NO: 154) and 15G11.92A (light chain—SEQ ID NO: 151 andheavy chain—SEQ ID NO: 108). The HVRs according to Kabat are indicatedby underlining.

FIG. 25 depicts a competition assay between 15G11v.5 and anti-TfR^(c12)as described in Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 1. Definitions

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., andantibody) and its binding partner (e.g., an antigen). Unless indictedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (KD, which is a ratio of the off-rate of X from Y (kd or Koff)to the on-rate of X to Y (ka or kon)). A surrogate measurement for theaffinity of one or more antibodies for its target is its half maximalinhibitory concentration (IC50), a measure of how much of the antibodyis needed to inhibit the binding of a known ligand to the antibodytarget by 50%. Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are describedherein. The “blood-brain barrier” or “BBB” refers to the physiologicalbarrier between the peripheral circulation and the brain and spinal cord(i.e., the CNS) which is formed by tight junctions within the braincapillary endothelial plasma membranes, creating a tight barrier thatrestricts the transport of molecules into the brain, even very smallmolecules such as urea (60 Daltons). The blood-brain barrier within thebrain, the blood-spinal cord barrier within the spinal cord, and theblood-retinal barrier within the retina are contiguous capillarybarriers within the CNS, and are herein collectively referred to a theblood-brain barrier or BBB. The BBB also encompasses the blood-CSFbarrier (choroid plexus) where the barrier is comprised of ependymalcells rather than capillary endothelial cells.

The terms “amyloid beta,” “beta-amyloid,” “Abeta,” “amyloidβ,” and “Aβ”,used interchangeably herein, refer to the fragment of amyloid precursorprotein (“APP”) that is produced upon β-secretase 1 (“BACE1”) cleavageof APP, as well as modifications, fragments and any functionalequivalents thereof, including, but not limited to, Aβ₁₋₄₀, and Aβ₁₋₄₂.Aβ is known to exist in monomeric form, as well as to associate to formoligomers and fibril structures, which may be found as constituentmembers of amyloid plaque. The structure and sequences of such Aβpeptides are well known to one of ordinary skill in the art and methodsof producing said peptides or of extracting them from brain and othertissues are described, for example, in Glenner and Wong, Biochem BiophysRes. Comm. 129: 885-890 (1984). Moreover, Aβ peptides are alsocommercially available in various forms.

“Anti-Abeta immunoglobulin,” “anti-Abeta antibody,” and “antibody thatbinds Abeta” are used interchangeably herein, and refer to an antibodythat specifically binds to human Abeta. A nonlimiting example of ananti-Abeta antibody is crenezumab. Other non-limiting examples ofanti-Abeta antibodies are solanezumab, bapineuzumab, gantenerumab,aducanumab, ponezumab and any anti-Abeta antibodies disclosed in thefollowing publications: WO2000162801, WO2002046237, WO2002003911,WO2003016466, WO2003016467, WO2003077858, WO2004029629, WO2004032868,WO2004032868, WO2004108895, WO2005028511, WO2006039470, WO2006036291,WO2006066089, WO2006066171, WO2006066049, WO2006095041, WO2009027105.

The terms “crenezumab” and “MABT5102A” are used interchangeably herein,and refer to a specific anti-Abeta antibody that binds to monomeric,oligomeric, and fibril forms of Abeta, and which is associated with CASregistry number 1095207. In some embodiments, such antibody comprisessequences set forth in FIGS. 18A and 18B.

“Apolipoprotein E4 carrier” or “ApoE4 carrier,” used interchangeablyherein with “apolipoprotein E4 positive” or “ApoE4 positive,” refers toan individual having at least one apolipoprotein E4 (or “ApoE4”) allele.An individual with zero ApoE4 alleles is referred to herein as being“ApoE4 negative” or an “ApoE4 non-carrier.” See also Prekumar, et al.,1996, Am. J Pathol. 148:2083-95.

The term “cerebral vasogenic edema” refers to an excess accumulation ofintravascular fluid or protein in the intracellular or extracellularspaces of the brain. Cerebral vasogenic edema is detectable by, e.g.,brain MRI, including, but not limited to FLAIR MRI, and can beasymptomatic (“asymptomatic vasogenic edema”) or associated withneurological symptoms, such as confusion, dizziness, vomiting, andlethargy (“symptomatic vasogenic edema”) (see Sperling et al.Alzheimer's & Dementia, 7:367, 2011).

The term “cerebral macrohemorrhage” refers to an intracranialhemorrhage, or bleeding in the brain, of an area that is more than about1 cm in diameter. Cerebral macrohemorrhage is detectable by, e.g., brainMRI, including but not limited to T2*-weighted GRE MRI, and can beasymptomatic (“asymptomatic macrohemorrhage”) or associated withsymptoms such as transient or permanent focal motor or sensoryimpairment, ataxia, aphasia, and dysarthria (“symptomaticmacrohemorrhage”) (see, e.g., Chalela J A, Gomes J. Expert Rev.Neurother. 2004 4:267, 2004 and Sperling et al. Alzheimer's & Dementia,7:367, 2011).

The term “cerebral microhemorrhage” refers to an intracranialhemorrhage, or bleeding in the brain, of an area that is less than about1 cm in diameter. Cerebral microhemorrhage is detectable by, e.g., brainMRI, including, but not limited to T2*-weighted GRE MRI, and can beasymptomatic (“asymptomatic microhemorrhage”) or can potentially beassociated with symptoms such as transient or permanent focal motor orsensory impairment, ataxia, aphasia, and dysarthria (“symptomaticmicrohemorrhage”). See, e.g., Greenberg, et al., 2009, Lancet Neurol.8:165-74.

The term “sulcal effusion” refers to an effusion of fluid in thefurrows, or sulci, of the brain. Sulcal effusions are detectable by,e.g., brain MRI, including but not limited to FLAIR MRI. See Sperling etal. Alzheimer's & Dementia, 7:367, 2011.

The term “superficial siderosis of the central nervous system” refers tobleeding or hemorrhage into the subarachnoid space of the brain and isdetectable by, e.g., brain MRI, including but not limited toT2*-weighted GRE MRI. Symptoms indicative of superficial siderosis ofthe central nervous system include sensorineural deafness, cerebellarataxia, and pyramidal signs. See Kumara-N, Am J Neuroradiol. 31:5, 2010.

The term “amyloidosis,” as used herein, refers to a group of diseasesand disorders caused by or associated with amyloid or amyloid-likeproteins and includes, but is not limited to, diseases and disorderscaused by the presence or activity of amyloid-like proteins inmonomeric, fibril, or polymeric state, or any combination of the three,including by amyloid plaques. Such diseases include, but are not limitedto, secondary amyloidosis and age-related amyloidosis, such as diseasesincluding, but not limited to, neurological disorders such asAlzheimer's Disease (“AD”), diseases or conditions characterized by aloss of cognitive memory capacity such as, for example, mild cognitiveimpairment (MCI), Lewy body dementia, Down's syndrome, hereditarycerebral hemorrhage with amyloidosis (Dutch type), the GuamParkinson-Demential complex and other diseases which are based on orassociated with amyloid-like proteins such as progressive supranuclearpalsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis),inclusion-body myositis (IBM), adult onset diabetes, endocrine tumor andsenile cardiac amyloidosis, and various eye diseases including maculardegeneration, drusen-related optic neuropathy, glaucoma, and cataractdue to beta-amyloid deposition.

Glaucoma is a group of diseases of the optic nerve involving loss ofretinal ganglion cells (RGCs) in a characteristic pattern of opticneuropathy. RGCs are the nerve cells that transmit visual signals fromthe eye to the brain. Caspase-3 and Caspase-8, two major enzymes in theapoptotic process, are activated in the process leading to apoptosis ofRGCs. Caspase-3 cleaves amyloid precursor protein (APP) to produceneurotoxic fragments, including Abeta. Without the protective effect ofAPP, Abeta accumulation in the retinal ganglion cell layer results inthe death of RGCs and irreversible loss of vision.

Glaucoma is often, but not always, accompanied by an increased eyepressure, which may be a result of blockage of the circulation ofaqueous, or its drainage. Although raised intraocular pressure is asignificant risk factor for developing glaucoma, no threshold ofintraocular pressure can be defined which would be determinative forcausing glaucoma. The damage may also be caused by poor blood supply tothe vital optic nerve fibers, a weakness in the structure of the nerve,and/or a problem in the health of the nerve fibers themselves. Untreatedglaucoma leads to permanent damage of the optic nerve and resultantvisual field loss, which can progress to blindness.

The term “mild Alzheimer's Disease” or “mild AD” as used herein (e.g., a“patient diagnosed with mild AD”) refers to a stage of AD characterizedby an MMSE score of 20 to 26. The term “mild to moderate Alzheimer'sDisease” or “mild to moderate AD” as used herein encompasses both mildand moderate AD, and is characterized by an MMSE score of 18 to 26.

The term “moderate Alzheimer's Disease” or “moderate AD” as used herein(e.g., a “patient diagnosed with moderate AD”) refers to a stage of ADcharacterized by an MMSE score of 18 to 19.

The “central nervous system” or “CNS” refers to the complex of nervetissues that control bodily function, and includes the brain and spinalcord.

A “blood-brain barrier receptor” (abbreviated “BBB-R” herein) is atransmembrane receptor protein expressed on brain endothelial cellswhich is capable of transporting molecules across the blood-brainbarrier. Examples of BBB-R include, but are not limited to: transferrinreceptor (TfR), insulin receptor, insulin-like growth factor receptor(IGF-R), low density lipoprotein receptors including without limitationlow density lipoprotein receptor-related protein 1 (LRP1) and lowdensity lipoprotein receptor-related protein 8 (LRP8), glucosetransporter 1 (Glut1) and heparin-binding epidermal growth factor-likegrowth factor (HB-EGF). An exemplary BBB-R herein is transferrinreceptor (TfR).

The term “transferrin receptor” or “TfR”, as used herein, refers to anynative TfR from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessed TfRas well as any form of TfR that results from processing in the cell. Theterm also encompasses naturally occurring variants of TfR, e.g., splicevariants or allelic variants. TfR is a transmembrane glycoprotein (witha molecular weight of about 180,000) composed of two disulphide-bondedsub-units (each of apparent molecular weight of about 90,000) involvedin iron uptake in vertebrates. In some embodiments, the TfR herein ishuman TfR (“hTfR”) comprising the amino acid sequence as set forth inSchneider et al. Nature 311: 675-678 (1984), for example (SEQ ID NO: 1).In another embodiment, the TfR herein is primate TfR (“pTfR”) comprisingthe amino acid sequence as set forth in Genbank reference AFD18260.1(SEQ ID NO: 2). For comparison, the mouse TfR sequence may be found inGenbank reference AAH54522.1 (SEQ ID NO: 3).

A “neurological disorder” as used herein refers to a disease or disorderwhich affects the CNS and/or which has an etiology in the CNS. ExemplaryCNS diseases or disorders include, but are not limited to, neuropathy,amyloidosis, cancer, an ocular disease or disorder, viral or microbialinfection, inflammation, ischemia, neurodegenerative disease, seizure,behavioral disorders, and a lysosomal storage disease. For the purposesof this application, the CNS will be understood to include the eye,which is normally sequestered from the rest of the body by theblood-retina barrier. Specific examples of neurological disordersinclude, but are not limited to, neurodegenerative diseases (including,but not limited to, Lewy body disease, postpoliomyelitis syndrome,Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease,multiple system atrophy, striatonigral degeneration, tauopathies(including, but not limited to, Alzheimer disease and supranuclearpalsy), prion diseases (including, but not limited to, bovine spongiformencephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), bulbar palsy, motor neuron disease, andnervous system heterodegenerative disorders (including, but not limitedto, Canavan disease, Huntington's disease, neuronalceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkeskinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome,lafora disease, Rett syndrome, hepatolenticular degeneration,Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia(including, but not limited to, Pick's disease, and spinocerebellarataxia), cancer (e.g. of the CNS, including brain metastases resultingfrom cancer elsewhere in the body).

A “neurological disorder drug” is a drug or therapeutic agent thattreats one or more neurological disorder(s). Neurological disorder drugsof the invention include, but are not limited to, antibodies, peptides,proteins, natural ligands of one or more CNS target(s), modifiedversions of natural ligands of one or more CNS target(s), aptamers,inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNA) and shorthairpin RNAs (shRNA)), ribozymes, and small molecules, or activefragments of any of the foregoing. Exemplary neurological disorder drugsof the invention are described herein and include, but are not limitedto: antibodies, aptamers, proteins, peptides, inhibitory nucleic acidsand small molecules and active fragments of any of the foregoing thateither are themselves or specifically recognize and/or act upon (i.e.,inhibit, activate, or detect) a CNS antigen or target molecule such as,but not limited to, amyloid precursor protein or portions thereof,amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein,parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancermarkers, and neurotrophins. Non-limiting examples of neurologicaldisorder drugs and the disorders they may be used to treat are providedin the following Table 1:

TABLE 1 Non-limiting examples of neurological disorder drugs and thecorresponding disorders they may be used to treat Drug Neurologicaldisorder Anti-BACE1 Antibody Alzheimer's, acute and chronic braininjury, stroke Anti-Abeta Antibody Alzheimer's disease Anti-Tau AntibodyAlzheimer's disease, tauopathies Neurotrophin Stroke, acute braininjury, spinal cord injury Brain-derived neurotrophic factor (BDNF),Chronic brain injury (Neurogenesis) Fibroblast growth factor 2 (FGF-2)Anti-Epidermal Growth Factor Receptor Brain cancer (EGFR)-antibody Glialcell-line derived neural factor (GDNF) Parkinson's disease Brain-derivedneurotrophic factor (BDNF) Amyotrophic lateral sclerosis, depressionLysosomal enzyme Lysosomal storage disorders of the brain Ciliaryneurotrophic factor (CNTF) Amyotrophic lateral sclerosis Neuregulin-1Schizophrenia Anti-HER2 antibody (e.g. trastuzumab, Brain metastasisfrom HER2-positive cancer pertuzumab, etc.) Anti-VEGF antibody (e.g.,bevacizumab) Recurrent or newly diagnosed glioblastoma, recurrentmalignant glioma, brain metastasis

An “imaging agent” is a compound that has one or more properties thatpermit its presence and/or location to be detected directly orindirectly. Examples of such imaging agents include proteins and smallmolecule compounds incorporating a labeled moiety that permitsdetection.

A “CNS antigen” or “brain antigen” is an antigen expressed in the CNS,including the brain, which can be targeted with an antibody or smallmolecule. Examples of such antigens include, without limitation:beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin6 receptor (IL6R), TNF receptor 1 (TNFR1), interleukin 1 beta (IL1β),and caspase 6. In some embodiments, the antigen is BACE1.

The term “BACE1,” as used herein, refers to any native beta-secretase 1(also called β-site amyloid precursor protein cleaving enzyme 1,membrane-associated aspartic protease 2, memapsin 2, aspartyl protease 2or Asp2) from any vertebrate source, including mammals such as primates(e.g. humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed BACE1 as wellas any form of BACE1 which results from processing in the cell. The termalso encompasses naturally occurring variants of BACE1, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryBACE1 polypeptide is the sequence for human BACE1, isoform A as reportedin Vassar et al., Science 286:735-741 (1999), which is incorporatedherein by reference in its entirety. Several other isoforms of humanBACE1 exist including isoforms B, C and D. See UniProtKB/Swiss-ProtEntry P56817, which is incorporated herein by reference in its entirety.

The terms “anti-beta-secretase antibody”, “anti-BACE1 antibody”, “anantibody that binds to beta-secretase” and “an antibody that binds toBACE1” refer to an antibody that is capable of binding BACE1 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting BACE1. In some embodiments, theextent of binding of an anti-BACE1 antibody to an unrelated, non-BACE1protein is less than about 10% of the binding of the antibody to BACE1as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to BACE1 has a dissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸Mor less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). Incertain embodiments, an anti-BACE1 antibody binds to an epitope of BACE1that is conserved among BACE1 from different species and isoforms. Insome embodiments, an antibody is provided that binds to the epitope onBACE1 bound by anti-BACE1 antibody YW412.8.31. In other embodiments, anantibody is provided that binds to an exosite within BACE1 located inthe catalytic domain of BACE1. In some embodiments an antibody isprovided that competes with the peptides identified in Kornacker et al.,Biochem. 44:11567-11573 (2005), which is incorporated herein byreference in its entirety, (i.e., Peptides 1, 2, 3, 1-11, 1-10, 1-9,1-8, 1-7, 1-6, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4,5, 6, 5-10, 5-9, scrambled, YSA, P6A, Y7A, F8A, I9A, P10A and L11A) forbinding to BACE1. Exemplary BACE1 antibody sequences are depicted inFIG. 15A-B and FIG. 16A-B. One exemplary antibody herein comprises thevariable domains of the antibody YW412.8.31 (e.g. as in FIGS. 15A-B).

A “native sequence” protein herein refers to a protein comprising theamino acid sequence of a protein found in nature, including naturallyoccurring variants of the protein. The term as used herein includes theprotein as isolated from a natural source thereof or as recombinantlyproduced.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments are well known in the art (see, e.g., Nelson, MAbs (2010)2(1): 77-83) and include but are not limited to Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv; diabodies; linear antibodies; single-chain antibodymolecules including but not limited to single-chain variable fragments(scFv), fusions of light and/or heavy-chain antigen-binding domains withor without a linker (and optionally in tandem); and monospecific ormultispecific antigen-binding molecules formed from antibody fragments(including, but not limited to multispecific antibodies constructed frommultiple variable domains which lack Fc regions).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants, e.g.,containing naturally occurring mutations or that may arise duringproduction of the monoclonal antibody, such variants generally beingpresent in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton the antigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including but not limited to thehybridoma method (see, e.g., Kohler et al., Nature, 256:495 (1975)),recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display methods (e.g., using the techniques described in Clacksonet al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991)), and methods utilizing transgenic animals containingall or part of the human immunoglobulin loci, such methods and otherexemplary methods for making monoclonal antibodies being describedherein. Specific examples of monoclonal antibodies herein includechimeric antibodies, humanized antibodies, and human antibodies,including antigen-binding fragments thereof.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. Insome embodiments, for the VL, the subgroup is subgroup kappa I as inKabat et al., supra. In some embodiments, for the VH, the subgroup issubgroup III as in Kabat et al., supra.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanantibodies. For the most part, humanized antibodies are human antibodies(recipient antibody) in which residues from a hypervariable region ofthe recipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.For example, in certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of theframework regions (FRs) correspond to those of a human antibody. In someinstances, FR residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human antibody and all or substantially allof the FRs are those of a human antibody, except for FR substitution(s)as noted above. The humanized antibody optionally also will comprise atleast a portion of an antibody constant region, typically that of ahuman antibody. A “humanized form” of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

A “human antibody” herein is an antibody comprising an amino acidsequence structure that corresponds with the amino acid sequencestructure of an antibody produced by a human or a human cell or derivedfrom a non-human source that utilizes human antibody repertoires orother human antibody-encoding sequences. This definition of a humanantibody specifically excludes a humanized antibody comprising non-humanantigen-binding residues. Such antibodies can be identified or made by avariety of techniques, including, but not limited to: production bytransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing human antibodies in the absence of endogenous immunoglobulinproduction (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos.5,591,669, 5,589,369 and 5,545,807)); selection from phage displaylibraries expressing human antibodies or human antibody fragments (see,for example, McCafferty et al., Nature 348:552-553 (1990); Johnson etal., Current Opinion in Structural Biology 3:564-571 (1993); Clackson etal., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597(1991); Griffith et al., EMBO J. 12:725-734 (1993); U.S. Pat. Nos.5,565,332 and 5,573,905); generation via in vitro activated B cells (seeU.S. Pat. Nos. 5,567,610 and 5,229,275); and isolation from humanantibody-producing hybridomas.

A “multispecific antibody” herein is an antibody having bindingspecificities for at least two different epitopes. Exemplarymultispecific antibodies may bind both a TfR and a brain antigen.Multispecific antibodies can be prepared as full-length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Engineeredantibodies with two, three or more (e.g. four) functional antigenbinding sites are also contemplated (see, e.g., US Appln No. US2002/0004587 A1, Miller et al.). Multispecific antibodies can beprepared as full length antibodies or as antibody fragments.

Antibodies herein include “amino acid sequence variants” with alteredantigen-binding or biological activity. Examples of such amino acidalterations include antibodies with enhanced affinity for antigen (e.g.“affinity matured” antibodies), and antibodies with altered Fc region,if present, e.g. with altered (increased or diminished) antibodydependent cellular cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) (see, for example, WO 00/42072, Presta, L. and WO99/51642, Iduosogie et al.); and/or increased or diminished serumhalf-life (see, for example, WO00/42072, Presta, L.).

An “affinity modified variant” has one or more substituted hypervariableregion or framework residues of a parent antibody (e.g. of a parentchimeric, humanized, or human antibody) that alter (increase or reduce)affinity. A convenient way for generating such substitutional variantsuses phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino substitutions ateach site. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e.g. binding affinity). In order to identify candidate hypervariableregion sites for modification, alanine scanning mutagenesis can beperformed to identify hypervariable region residues contributingsignificantly to antigen binding. Alternatively, or additionally, it maybe beneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and its target.Such contact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningand antibodies with altered affinity may be selected for furtherdevelopment.

A “pH-sensitive antibody variant” is an antibody variant which has adifferent binding binding affinity for a target antigen at a first pHthan it does for that target antigen at a different pH. As a nonlimitingexample, an anti-TfR antibody of the invention may be selected for orengineered to have pH-sensitive binding to TfR such that it binds withdesirably low affinity (as described herein) to cell surface TfR in theplasma at pH 7.4, but upon internalization into an endosomalcompartment, rapidly dissociates from TfR at the relatively lower pH (pH5.5-6.0); such dissociation may protect the antibody fromantigen-mediated clearance, and increase the amount of antibody that iseither delivered to the CNS or recycled back across the BBB—in eithercase, the effective concentration of the antibody is increased relativeto an anti-TfR antibody that does not comprise such pH sensitivity (see,e.g., Chaparro-Riggers et al. J. Biol. Chem. 287(14): 11090-11097; Igawaet al., Nature Biotechnol. 28(11): 1203-1208). The desired combinationof affinities at the serum pH and the endosomal compartment pH can bereadily determined for a TfR and conjugated compound by one of ordinaryskill in the art.

The antibody herein may be conjugated with a “heterologous molecule” forexample to increase half-life or stability or otherwise improve theantibody. For example, the antibody may be linked to one of a variety ofnon-proteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. Antibody fragments, such as Fab′,linked to one or more PEG molecules are an exemplary embodiment of theinvention. In another example, the heterologous molecule is atherapeutic compound or a visualization agent (ie., a detectable label),and the antibody is being used to transport such heterologous moleculeacross the BBB. Examples of heterologous molecules include, but are notlimited to, a chemical compound, a peptide, a polymer, a lipid, anucleic acid, and a protein.

The antibody herein may be a “glycosylation variant” such that anycarbohydrate attached to the Fc region, if present, is altered, eithermodified in presence/absence, or modified in type. For example,antibodies with a mature carbohydrate structure that lacks fucoseattached to an Fc region of the antibody are described in US Pat Appl NoUS 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa HakkoKogyo Co., Ltd). Antibodies with a bisecting N-acetylglucosamine(GlcNAc) in the carbohydrate attached to an Fc region of the antibodyare referenced in WO 2003/011878, Jean-Mairet et al. and U.S. Pat. No.6,602,684, Umana et al. Antibodies with at least one galactose residuein the oligosaccharide attached to an Fc region of the antibody arereported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju,S.) and WO 1999/22764 (Raju, S.) concerning antibodies with alteredcarbohydrate attached to the Fc region thereof. See also US 2005/0123546(Umana et al.) describing antibodies with modified glycosylation.Mutation of the consensus glycosylation sequence in the Fc region(Asn-X-Ser/Thr at positions 297-299, where X cannot be proline), forexample by mutating the Asn of this sequence to any other amino acid, byplacing a Pro at position 298, or by modifying position 299 to any aminoacid other than Ser or Thr should abrogate glycosylation at thatposition (see, e.g., Fares Al-Ejeh et al., Clin. Cancer Res. (2007)13:5519s-5527s; Imperiali and Shannon, Biochemistry (1991) 30(18):4374-4380; Katsuri, Biochem J. (1997) 323(Pt 2): 415-419;Shakin-Eshleman et al., J. Biol. Chem. (1996) 271: 6363-6366).

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contact”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

In some embodiments, HVR residues comprise those identified in FIGS.3A-D or 4A-D, Table 4 or Table 5 or elsewhere in the specification.

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3 andFR4. Accordingly, the HVR and FR sequences generally appear in thefollowing sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.In certain embodiments, one or more FR residue may be modified tomodulate the stability of the antibody or to modulate thethree-dimensional positioning of one or more HVR of the antibody to,e.g., enhance binding.

A “full length antibody” is one which comprises an antigen-bindingvariable region as well as a light chain constant domain (CL) and heavychain constant domains, CH1, CH2 and CH3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety or radiolabel). The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2 and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

Antibody “effector functions” refer to those biological activities of anantibody that result in activation of the immune system other thanactivation of the complement pathway. Such activities are largely foundin the Fc region (a native sequence Fc region or amino acid sequencevariant Fc region) of an antibody. Examples of antibody effectorfunctions include, for example, Fc receptor binding andantibody-dependent cell-mediated cytotoxicity (ADCC). In someembodiments, the antibody herein essentially lacks effector function. Inanother embodiment, the antibody herein retains minimal effectorfunction. Methods of modifying or eliminating effector function arewell-known in the art and include, but are not limited to, eliminatingall or a portion of the Fc region responsible for the effector function(ie, using an antibody or antibody fragment in a format lacking all or aportion of the Fc region such as, but not limited to, a Fab fragment, asingle-chain antibody, and the like as described herein and as known inthe art; modifying the Fc region at one or more amino acid positions toeliminate effector function (Fc binding-impacting: positions 238, 239,248, 249, 252, 254, 256, 265, 268, 269, 270, 272, 278, 289, 292, 293,294, 295, 296, 297, 298, 301, 303, 311, 322, 324, 327, 329, 333, 335,338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 436, 437,438, and 439; and modifying the glycosylation of the antibody(including, but not limited to, producing the antibody in an environmentthat does not permit wild-type mammalian glycosylation, removing one ormore carbohydrate groups from an already-glycosylated antibody, andmodifying the antibody at one or more amino acid positions to eliminatethe ability of the antibody to be glycosylated at those positions(including, but not limited to N297G and N297A and D265A).

Antibody “complement activation” functions, or properties of an antibodythat enable or trigger “activation of the complement pathway” are usedinterchangeably, and refer to those biological activities of an antibodythat engage or stimulate the complement pathway of the immune system ina subject. Such activities include, e.g., C1q binding and complementdependent cytotoxicity (CDC), and may be mediated by both the Fc portionand the non-Fc portion of the antibody. Methods of modifying oreliminating complement activation function are well-known in the art andinclude, but are not limited to, eliminating all or a portion of the Fcregion responsible for complement activation (ie., using an antibody orantibody fragment in a format lacking all or a portion of the Fc regionsuch as, but not limited to, a Fab fragment, a single-chain antibody,and the like as described herein and as known in the art, or modifyingthe Fc region at one or more amino acid positions to eliminate or lesseninteractions with complement components or the ability to activatecomplement components, such as positions 270, 322, 329 and 321, known tobe involved in C1q binding), and modifying or eliminating a portion ofthe non-Fc region responsible for complement activation (ie, modifyingthe CH1 region at position 132 (see, e.g., Vidarte et al., (2001) J.Biol. Chem. 276(41): 38217-38223)).

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known inthe art.

The term “recombinant antibody”, as used herein, refers to an antibody(e.g. a chimeric, humanized, or human antibody or antigen-bindingfragment thereof) that is expressed by a recombinant host cellcomprising nucleic acid encoding the antibody.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cells and progeny derived therefrom without regardto the number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.Examples of “host cells” for producing recombinant antibodies include:(1) mammalian cells, for example, Chinese Hamster Ovary (CHO), COS,myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK),Hela and Vero cells; (2) insect cells, for example, sf9, sf21 and Tn5;(3) plant cells, for example plants belonging to the genus Nicotiana(e.g. Nicotiana tabacum); (4) yeast cells, for example, those belongingto the genus Saccharomyces (e.g. Saccharomyces cerevisiae) or the genusAspergillus (e.g. Aspergillus niger); (5) bacterial cells, for exampleEscherichia coli cells or Bacillus subtilis cells, etc.

As used herein, “specifically binding” or “binds specifically to” refersto an antibody selectively or preferentially binding to an antigen. Thebinding affinity is generally determined using a standard assay, such asScatchard analysis, or surface plasmon resonance technique (e.g. usingBIACORE®).

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. In some embodiments, an anti-BACE1antibody forming one of the bispecific or multispecific antibodies ofthe invention binds to the BACE1 epitope bound by YW412.8.31. Anexemplary competition assay is provided herein.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Rc¹⁸⁶, Rc¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed herein.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In some embodiments, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. Unless otherwise specified herein,numbering of amino acid residues in the Fc region or constant region isaccording to the EU numbering system, also called the EU index, asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991.

The term “FcRn receptor” or “FcRn” as used herein refers to an Fcreceptor (“n” indicates neonatal) which is known to be involved intransfer of maternal IgGs to a fetus through the human or primateplacenta, or yolk sac (rabbits) and to a neonate from the colostrumthrough the small intestine. It is also known that FcRn is involved inthe maintenance of constant serum IgG levels by binding the IgGmolecules and recycling them into the serum. “FcRn binding region” or“FcRn receptor binding region” refers to that portion an antibody whichinteracts with the FcRn receptor. Certain modifications in the FcRnbinding region of an antibody increase the affinity of the antibody orfragment thereof, for the FcRn, and also increase the in vivo half-lifeof the molecule. Amino acid substitutions in one or more of thefollowing amino acid positions 251 256, 285, 290, 308, 314, 385, 389,428, 434 and 436 increases the interaction of the antibody with the FcRnreceptor. Substitutions at the following positions also increases theinteraction of an antibody with the FcRn receptor 238, 265, 272, 286,303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382,413, 424 or 434, e.g., substitution of (U.S. Pat. No. 7,371,826).

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a label orcytotoxic agent. Optionally such conjugation is via a linker.

A “linker” as used herein is a structure that covalently ornon-covalently connects the anti-TfR antibody to heterologous molecule.In certain embodiments, a linker is a peptide. In other embodiments, alinker is a chemical linker.

A “label” is a marker coupled with the antibody herein and used fordetection or imaging. Examples of such labels include: radiolabel, afluorophore, a chromophore, or an affinity tag. In some embodiments, thelabel is a radiolabel used for medical imaging, for example tc99m or1123, or a spin label for nuclear magnetic resonance (NMR) imaging (alsoknown as magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese, iron, etc.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC) methods. For review of methods for assessment of antibody purity,see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-TfR antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject., A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W. H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

2. Compositions and Methods

A. Production of Anti-TfR Antibodies and Conjugates Thereof

In some aspects, the invention is based, in part, on anti-TfR antibodiesthat can be used to transport desired molecules across the BBB. Incertain embodiments, antibodies that bind to human TfR are provided. Incertain embodiments, antibodies that bind to both human TfR and primateTfR are provided. Antibodies of the invention are useful, e.g., for thediagnosis or treatment of diseases affecting the brain and/or CNS.

A. Exemplary Anti-TfR Antibodies

In some aspects, the invention provides isolated antibodies that bind toTfR. In certain embodiments, an anti-TfR antibody of the invention bindsspecifically to both human TfR and primate TfR. In certain suchembodiments, an anti-TfR antibody of the invention does not inhibitbinding of transferrin to the TfR. In certain such embodiments, ananti-TfR antibody of the invention binds to an apical domain of TfR. Inother certain such embodiments, an anti-TfR antibody of the inventionbinds to a non-apical domain of TfR. In certain aspects, the anti-TfRantibodies may be used to transport one or more conjugated imaging ortherapeutic compounds across the BBB.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:32; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:33; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:34; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:29; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:31. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 7A4, as shown in FIG. 3A and Table3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:38; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:39; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:35; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:36. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 8A2, as shown in FIG. 3A and Table3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:40; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 36. In some aspects,the antibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 15D2, as shown in FIG. 3A andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 43; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 44; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 42. In some aspects,the antibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 10D11, as shown in FIG. 3A andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:31. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 7B10, as shown in FIG. 3A andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:53; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:54; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:55; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:50; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:51; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:52. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 15G11, as shown in FIG. 3B andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:53; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:58; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:59; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:56; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:57; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:52. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 16G5, as shown in FIG. 3B andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:53; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:63; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:55; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:60; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:61; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:62. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 13C3, as shown in FIG. 3B andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:53; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:65; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:55; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:60; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:64; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:52. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 16G4, as shown in FIG. 3B andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:74; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:75; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:76; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:71; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:72; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:73. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 16F6, as shown in FIG. 3C andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:80; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:81; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:82; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:77; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:78; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:79. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 7G7, as shown in FIG. 3D and Table3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 80; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:83; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:84; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:77; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:78; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:79. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 4C2, as shown in FIG. 3D and Table3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:88; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:89; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:90; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:85; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:86; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:87. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 1B12, as shown in FIG. 3D andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:94; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:95; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:96; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:91; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:92; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:93. In some aspects, theantibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 13D4, as shown in FIG. 3D andTable 3.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:32; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:33; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:34; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:29; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:127. In some aspects,the antibody comprises all six of the above-recited HVR sequences. Inanother aspect, the antibody is clone 7A4.v15, as shown in FIG. 4B andTable 4.

The clones above fall into four complementation groups, with sequencesimilarity within the HVRs. As shown in Table 3, consensus sequences arereadily derivable from the provided antibody sequences for each HVR. Asone nonlimiting example, the class I antibody consensus HVRs are asfollows:

HVR-L1: (SEQ ID NO: 45)Arg-Ala-Ser-Glu-Ser-Val-Asp-[Ser or Asp]-Tyr-Gly-[Asn or Pro]-Ser-Phe-Met-His; HVR-L2: (SEQ ID NO: 30)Arg-Ala-Ser-Asn-Leu-Glu-Ser; HVR-L3: (SEQ ID NO: 46)Gln-[Gln or His]-Ser-Asn-Glu-[Ala, Gly or Asp]- Pro-Pro-Thr; HVR-H1:(SEQ ID NO: 47) Asp-Tyr-[Ala or Gly]-Met-His; HVR-H2: (SEQ ID NO: 48)[Gly or Val]-Ile-Ser-[Thr, Phe or Pro]-Tyr-[Phe or Ser]-Gly-[Arg or Lys]-Thr-Asn-Tyr-[Asn or Ser]-Gln-[Lys or Asn]-Phe-[Lys or Met]- Gly; HVR-H3:(SEQ ID NO: 49) Gly-Leu-Ser-Gly-Asn-[Tyr or Phe]-Val-[Met orVal]-Asp-[Thr or Phe].(see Table 4). The consensus sequences for class II and IV are alsoprovided in Table 4.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:47; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:48; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:49; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:45; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:30; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:46. In some aspects, theantibody comprises all six of the above-recited HVR sequences.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:53; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:69; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:70; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:66; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:67; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:68. In some aspects, theantibody comprises all six of the above-recited HVR sequences.

In some aspects, an anti-TfR antibody is provided comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:100; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:101; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:97; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:98; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:99. In some aspects, theantibody comprises all six of the above-recited HVR sequences.

In some aspects, an antibody is provided comprising at least one, atleast two, or all three VH HVR sequences of any of the antibodiesdescribed above. In some embodiments, the antibody comprises the HVR-H3sequence of any one of the antibodies described above. In anotherembodiment, the antibody comprises the HVR-H3 and HVR-L3 sequences ofany one of the antibodies described above. In a further embodiment, theantibody comprises the HVR-H3, HVR-L3 and HVR-H2 sequences of any one ofthe antibodies described above. In another embodiment, the antibodycomprises the HVR-H1, HVR-H2 and HVR-H3 sequences of any one of theantibodies described above. In another aspect, an antibody is providedcomprising at least one, at least two or all three VL HVR sequences ofany of the antibodies described above. In some embodiments, the antibodycomprises the HVR-L1, HVR-L2, and HVR-L3 sequences of any one of theantibodies described above.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from the HVR-H1, HVR-H2, and HVR-H3 sequences of anyone of the antibodies described above, wherein the VH domain comprisesan M108L mutation; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from the HVR-L1, HVR-L2 andHVR-L3 sequences of any one of the antibodies described above.

In any of the above embodiments, an anti-TfR antibody is humanized. Insome embodiments, an anti-TfR antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework. Inanother embodiment, an anti-TfR antibody comprises HVRs as in any of theabove embodiments, and further comprises a VH or VL comprising one ormore amino acid substitutions in one or more FR regions. Per Example 2herein, Applicants performed alanine scanning on certain antibodiesselected from those above, and determined that similar or improvedbinding was obtained despite amino acid modifications at selected FRpositions. As shown in FIGS. 6-1 and 6-2 and Example 2 herein, for theclass I-III groups of antibodies, variant forms of the antibodies withmodifications at one or more residues of an FR retained affinity andbinding specifity. For example, for antibody 15G11, positions 43 and 48in the light chain FR2, position 48 in the heavy chain FR2 and positions67, 69, 71 and 73 in the heavy chain FR3 could be modified as shown inFIGS. 6-1 and 6-2 and the resulting antibody still retained specificityand strong binding affinity for human/primate TfR. In another example,for antibody 7A4, positions 58 and 68 of the light chain FR3, position24 in the heavy chain FR1 and position 71 in the heavy chain FR3 couldbe modified as shown in FIGS. 6-1 and 6-2 and the resulting antibodystill retained specificity and strong binding affinity for human/primateTfR. In a third example, for antibody 16F6, positions 43 and 44 of thelight chain FR2 and positions 71 and 78 of the heavy chain FR3 could bemodified as shown in FIGS. 6-1 and 6-2 and the resulting antibody stillretained specificity and strong binding affinity for human/primate TfR.In some embodiments, an antibody comprises an VH domain with an M108Lmutation.

In another aspect, an anti-TfR antibody comprises a heavy chain variabledomain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofany one of SEQ ID NOs: 7-10, 15-18, 20, 25-28, 108, 114, 120, 126, 153,and 154. In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-TfR antibodycomprising that sequence retains the ability to bind to TfR. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in any one of SEQ ID NOs:7-10, 15-18, 20, 25-28,108, 114, 120, 126, 153, and 154. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-TfR antibody comprises the VH sequence of anyone of SEQ ID NOs: 7-10, 15-18, 20 25-28, 108, 114, 120, 126, 153, and154, including post-translational modifications of that sequence. In aparticular embodiment, the VH for a particular antibody comprises one,two or three HVRs selected from: the HVRs set forth above and in Table 3or 4 for that particular antibody. VH sequences for certain antibodiesare shown in FIGS. 3 and 4 herein. In some embodiments, the inventionprovides an anti-TfR antibody comprising a VH sequence of SEQ ID NO: 158or 159.

In another aspect, an anti-TfR antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs:4-6, 11-14,19, 21-24, 105, 111, 117, 123, 151, and 152. In certain embodiments, aVL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-TfR antibody comprising that sequence retains theability to bind to TfR. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in any one of SEQID NOs:4-6, 11-14, 19, 21-24, 105, 111, 117, 123, 151, and 152. Incertain embodiments, the substitutions, insertions, or deletions occurin regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TfRantibody comprises the VL sequence in any of SEQ ID NOs:4-6, 11-14, 19,21-24, 105, 111, 117, 123, 151, and 152, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from the HVRs set forth aboveand in Table 4 or 5 for that particular antibody. VL sequences forcertain antibodies are shown in FIGS. 3 and 4 herein. In someembodiments, the invention provides an anti-TfR antibody comprising a VLsequence of SEQ ID NO: 162 or 163.

In another aspect, an anti-TfR antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In some embodiments,the antibody comprises the VL and VH sequences, respectively, in SEQ IDNOs: 4 and 7; 5 and 8; 5 and 9; 6 and 10; 4 and 7; 11 and 15; 12 and 16;13 and 17; 14 and 18; 19 and 20; 21 and 25; 22 and 26; 23 and 27; 24 and28; 105 and 108; 152 and 108; 151 and 108; 152 and 153; 152 and 154; 105and 153; 105 and 154; 111 and 114; 117 and 120; and 123 and 126,including post-translational modifications of those sequences. In someembodiments, the invention provides an anti-TfR antibody comprising a VHsequence of SEQ ID NO: 158 or 159 and a VL sequence of SEQ ID NO: 162 or163. In some embodiments, the invention provides an anti-TfR antibodycomprising a VH sequence of SEQ ID NO: 158 and a VL sequence of SEQ IDNO: 162, or a VH sequence of SEQ ID NO: 159 and a VL sequence of SEQ IDNO: 163.

In a further aspect, an antibody is provided that binds to the sameepitope as an anti-TfR antibody provided herein. For example, in certainembodiments, an antibody is provided that binds to the same epitope asan anti-TfR antibody comprising VL and VH sequences, respectively, ofSEQ ID NOs: 4 and 7; 5 and 8; 5 and 9; 6 and 10; 4 and 7; 11 and 15; 12and 16; 13 and 17; 14 and 18; 19 and 20; 21 and 25; 22 and 26; 23 and27; 24 and 28; 105 and 108; 152 and 108; 151 and 108; 152 and 153; 152and 154; 105 and 153; 105 and 154; 111 and 114; 117 and 120; or 123 and126. In some aspects, the antibody competes with any of the antibodiesin Class I (ie., clones 7A4, 8A2, 15D2, 10D11, or 7B10, oraffinity-matured versions of any of those antibodies) for binding toTfR. In another aspect, the antibody competes with any of the antibodiesin Class II (ie, clones 15G11, 16G5, 13C3 or 16G, or affinity-maturedversions of any of those antibodies) for binding to TfR. In anotheraspect, the antibody competes with clone 16F6 or affinity-maturedversions thereof for binding to TfR. In another aspect, the antibodycompetes with any of the antibodies in Class IV (ie, clones 7G7, 4C2,1B12 or 13D4, or affinity matured versions thereof) for binding to TfR.

In a further aspect of the invention, an anti-TfR antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In some embodiments, an anti-TfRantibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)2 fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1, IgG2, IgG3, or IgG4 antibody orother antibody class or isotype as defined herein.

In some embodiments, the invention provides an anti-TfR antibodycomprising a heavy chain having the sequence of SEQ ID NO: 160 and alight chain having the sequence of SEQ ID NO: 61. In some suchembodiments, the antibody is a multispecific antibody.

In a further aspect, an anti-TfR antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M).

In certain aspects of the present invention, a “low affinity” anti-TfRantibody of the invention is selected, based, e.g., on the results inExample 5 and in Atwal et al., Sci. Transl. Med. 3, 84ra43 (2011) and Yuet al., Sci. Transl. Med. 25 May 2011: Vol. 3, Issue 84, p. 84ra44,showing that such lower-affinity antibodies to TfR display improved CNS(for example, brain) uptake and/or persistence in the brain/CNS. Inorder to identify such low affinity antibodies, various assays formeasuring antibody affinity are available including, without limitation:Scatchard assay and surface plasmon resonance technique (e.g. usingBIACOREO). According to some embodiments of the invention, the antibodyhas an affinity for human or primate TfR from about 5 nM, or from about20 nM, or from about 100 nM, to about 50 μM, or to about 30 μM, or toabout 10 μM, or to about 1 μM, or to about 500 nM. Thus, the affinitymay be in the range from about 5 nM to about 50 μM, or in the range fromabout 20 nM to about 30 μM, or in the range from about 30 nM to about 30μM, or in the range from about 50 nM to about 1 μM, or in the range fromabout 100 nM to about 500 nM, e.g. as measured by Scatchard analysis orBIACORE®. In another embodiment of the invention, the antibody has adissociation half-life from TfR of less than 1 minute, less than 2minutes, less than 3 minutes, less than four minutes, less than 5minutes, or less than 10 minutes to about 20 minutes, or to about 30minutes, as measured by competition binding analysis or BIACORE®.

Thus, the invention provides a method of making an antibody useful fortransporting a neurological disorder drug across the blood-brain barriercomprising selecting an antibody from a panel of antibodies against TfRbecause it has an affinity for TfR which is in the range from about 5nM, or from about 20 nM, or from about 100 nM, to about 50 μM, or toabout 30 μM, or to about 10 μM, or to about 1 μM, or to about 500 mM.Thus, the affinity may be in the range from about 5 nM to about 50 μM,or in the range from about 20 nM to about 30 μM, or in the range fromabout 30 nM to about 30 μM, or in the range from about 50 nM to about 1μM, or in the range from about 100 nM to about 500 nM, e.g. as measuredby Scatchard analysis or BIACORE®. As will be understood by one ofordinary skill in the art, conjugating a heterologous molecule/compoundto an antibody will often decrease the affinity of the antibody for itstarget due, e.g., to steric hindrance or even to elimination of onebinding arm if the antibody is made multispecific with one or more armsbinding to a different antigen than the antibody's original target. Insome embodiments, a low affinity antibody of the invention specific forTfR conjugated to anti-BACE1 had a Kd for TfR as measured by BIACORE ofabout 30 nM. In another embodiment, a low affinity antibody of theinvention specific for TfR conjugated to BACE1 had a Kd for TfR asmeasured by BIACORE of about 600 nM. In another embodiment, a lowaffinity antibody of the invention specific for TfR conjugated to BACE1had a Kd for TfR as measured by BIACORE of about 20 μM. In anotherembodiment, a low affinity antibody of the invention specific for TfRconjugated to BACE1 had a Kd for TfR as measured by BIACORE of about 30μM.

In some embodiments, Kd is measured by a radiolabeled antigen bindingassay (RIA). In some embodiments, an RIA is performed with the Fabversion of an antibody of interest and its antigen. For example,solution binding affinity of Fabs for antigen is measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establishconditions for the assay, MICROTITER® multi-well plates (ThermoScientific) are coated overnight with 5 μg/ml of a capturing anti-Fabantibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), andsubsequently blocked with 2% (w/v) bovine serum albumin in PBS for twoto five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

In some aspects, the RIA is a Scatchard analysis. For example, theanti-TfR antibody of interest can be iodinated using the lactoperoxidasemethod (Bennett and Horuk, Methods in Enzymology 288 pg. 134-148(1997)). A radiolabeled anti-TfR antibody is purified from free ¹²⁵I-Naby gel filtration using a NAP-5 column and its specific activitymeasured. Competition reaction mixtures of 50 μL containing a fixedconcentration of iodinated antibody and decreasing concentrations ofserially diluted unlabeled antibody are placed into 96-well plates.Cells transiently expressing TfR are cultured in growth media,consisting of Dulbecco's modified eagle's medium (DMEM) (Genentech)supplemented with 10% FBS, 2 mM L-glutamine and1×penicillin-streptomycin at 37° C. in 5% CO₂. Cells are detached fromthe dishes using Sigma Cell Dissociation Solution and washed withbinding buffer (DMEM with 1% bovine serum albumin, 50 mM HEPES, pH 7.2,and 0.2% sodium azide). The washed cells are added at an approximatedensity of 200,000 cells in 0.2 mL of binding buffer to the 96-wellplates containing the 50-μL competition reaction mixtures. The finalconcentration of the unlabeled antibody in the competition reaction withcells is varied, starting at 1000 nM and then decreasing by 1:2 folddilution for 10 concentrations and including a zero-added, buffer-onlysample. Competition reactions with cells for each concentration ofunlabeled antibody are assayed in triplicate. Competition reactions withcells are incubated for 2 hours at room temperature. After the 2-hourincubation, the competition reactions are transferred to a filter plateand washed four times with binding buffer to separate free from boundiodinated antibody. The filters are counted by gamma counter and thebinding data are evaluated using the fitting algorithm of Munson andRodbard (1980) to determine the binding affinity of the antibody.

An exemplary BIACOREO analysis using the compositions of the inventionmay be performed as follows. Kd was measured using surface plasmonresonance assays using a BIACORE®-2000 (BlAcore, Inc., Piscataway, N.J.)at 25° C. using anti-human Fc kit (BiAcore Inc., Piscataway, N.J.).Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.)were activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Anti-human Fc antibody was diluted with 10 mMsodium acetate, pH 4.0, to 50 μg/ml before injection at a flow rate of 5μl/minute to achieve approximately 10000 response units (RU) of coupledprotein. Following the injection of antibody, 1 M ethanolamine wasinjected to block unreacted groups. For kinetics measurements,monospecific or multispecific anti-TfR antibody variants were injectedin HBS-P to reach about 220 RU, then two-fold serial dilutions ofMuTfR-His (0.61 nM to 157 nM) were injected in HBS-P at 25° C. at a flowrate of approximately 30 μl/min. Association rates (kon) anddissociation rates (koff) were calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) was calculated as the ratiokoff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)

According to another embodiment, Kd is measured using surface plasmonresonance assays with a BIACORE®-2000 device (BlAcore, Inc., Piscataway,N.J.) at 25° C. using anti-human Fc kit (BiAcore Inc., Piscataway,N.J.). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Anti-human Fc antibody is diluted with 10 mMsodium acetate, pH 4.0, to 50 μg/ml before injection at a flow rate of 5μl/minute to achieve approximately 10000 response units (RU) of coupledprotein. Following the injection of antibody, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, anti-TfRantibody variants are injected in HBS-P to reach about 220 RU, thentwo-fold serial dilutions of TfR-His (0.61 nM to 157 nM) are injected inHBS-P at 25° C. at a flow rate of approximately 30 μl/min. Associationrates (kon) and dissociation rates (koff) are calculated using a simpleone-to-one Langmuir binding model (BIACORE® Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (Kd) is calculated asthe ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999).

Several methods of determining the IC50 for a given compound areart-known; a common approach is to perform a competition binding assay,such as that described herein. In general, a high IC50 indicates thatmore of the antibody is required to inhibit binding of the known ligand,and thus that the antibody's affinity for that ligand is relatively low.Conversely, a low IC50 indicates that less of the antibody is requiredto inhibit binding of the known ligand, and thus that the antibody'saffinity for that ligand is relatively high.

An exemplary competitive ELISA assay to measure IC50 is one in whichincreasing concentrations of anti-TfR or anti-TfR/brain antigen (i.e.,anti-TfR/BACE1, anti-TfR/Abeta and the like) variant antibodies are usedto compete against a biotinylated known anti-TfR antibody for binding toTfR. The anti-TfR competition ELISA was performed in Maxisorp plates(Neptune, N.J.) coated with 2.5 μg/ml of purified murine TfRextracellular domain in PBS at 4° C. overnight. Plates were washed withPBS/0.05% Tween 20 and blocked using Superblock blocking buffer in PBS(Thermo Scientific, Hudson, N.H.). A titration of each individualanti-TfR or anti-TfR/brain antigen (i.e., anti-TfR/BACE1 oranti-TfR/Abeta) (1:3 serial dilution) was combined with biotinylatedknown anti-TfR (0.5 nM final concentration) and added to the plate for 1hour at room temperature. Plates were washed with PBS/0.05% Tween 20,and HRP-streptavidin (Southern Biotech, Birmingham) was added to theplate and incubated for 1 hour at room temperature. Plates were washedwith PBS/0.05% Tween 20, and biotinylated anti-TfR antibody bound to theplate was detected using TMB substrate (BioFX Laboratories, OwingsMills).

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, N.Y., pp. 269-315 (1994);see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Fordiscussion of Fab and F(ab′)₂ fragments comprising salvage receptorbinding epitope residues and having increased in vivo half-life, seeU.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. Chimeric antibodies of interest herein include“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g. Old World Monkey, suchas baboon, rhesus or cynomolgus monkey) and human constant regionsequences (U.S. Pat. No. 5,693,780). In a further example, a chimericantibody is a “class switched” antibody in which the class or subclasshas been changed from that of the parent antibody. Chimeric antibodiesinclude antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci USA, 103:3557-3562 (2006).Additional methods include those described, for example, in U.S. Pat.No. 7,189,826 (describing production of monoclonal human IgM antibodiesfrom hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology(Trioma technology) is also described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for TfR and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of TfR. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express TfR. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tuft et al. J.Immunol. 147: 60 (1991).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to TfR as well asanother, different antigen (see, US 2008/0069820, for example).

According to some embodiments of the invention, the “coupling” isachieved by generating a multispecific antibody (e.g. a bispecificantibody). Multispecific antibodies are monoclonal antibodies that havebinding specificities for at least two different antigens or epitopes.In some embodiments, the multispecific antibody comprises a firstantigen binding site which binds the TfR and a second antigen bindingsite which binds a brain antigen, such as beta-secretase 1 (BACE1) orAbeta, and the other brain antigens disclosed herein.

An exemplary brain antigen bound by such multispecific/bispecificantibody is BACE1, and an exemplary antibody binding thereto is theYW412.8.31 antibody in FIGS. 16A-B herein.

In another embodiment, the brain antigen is Abeta, exemplary suchantibodies being described in WO2007068412, WO2008011348, WO20080156622,and WO2008156621, expressly incorporated herein by reference, with anexemplary Abeta antibody comprising the IgG4 MABT5102A antibodycomprising the heavy and light chain amino acid sequences in FIGS. 11Aand 11B, respectively.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tuft et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs) are also included herein (see, e.g. US2006/0025576A1, and Wu et al. Nature Biotechnology (2007)).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 2 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 2 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e. g. complex,hybrid and high mannose structures) as measured by MALDI-TOF massspectrometry, as described in WO 2008/077546, for example. Asn297 refersto the asparagine residue located at about position 297 in the Fc region(Eu numbering of Fc region residues); however, Asn297 may also belocated about ±3 amino acids upstream or downstream of position 297,i.e., between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769(2006)).

Non-limiting examples of antibodies with reduced effector functioninclude those with substitution of one or more of Fc region residues238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such, non-limiting, Fc variants include those withsubstitutions at one or more of Fc region residues: 238, 256, 265, 272,286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S.Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In some embodiments, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In some embodiments,isolated nucleic acid encoding an anti-TfR antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In some embodiments, thehost cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a methodof making an anti-TfR antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-TfR antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-TfR antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

Various techniques are available for determining binding of the antibodyto the TfR. One such assay is an enzyme linked immunosorbent assay(ELISA) for confirming an ability to bind to human TfR (and brainantigen). According to this assay, plates coated with antigen (e.g.recombinant TfR) are incubated with a sample comprising the anti-TfRantibody and binding of the antibody to the antigen of interest isdetermined.

In some aspects, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies of the invention forbinding to TfR. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by any of the antibodies of the invention, more specifically, anyof the epitopes specifically bound by antibodies in class I, class II,class III or class IV as described herein (see, e.g., Example 1 andTable 4. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In an exemplary competition assay, immobilized TfR is incubated in asolution comprising a first labeled antibody that binds to TfR (e.g.,one or more of the antibodies disclosed herein) and a second unlabeledantibody that is being tested for its ability to compete with the firstantibody for binding to TfR. The second antibody may be present in ahybridoma supernatant. As a control, immobilized TfR is incubated in asolution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to TfR, excess unbound antibody isremoved, and the amount of label associated with immobilized TfR ismeasured. If the amount of label associated with immobilized TfR issubstantially reduced in the test sample relative to the control sample,then that indicates that the second antibody is competing with the firstantibody for binding to TfR. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

2. Activity Assays

In some aspects, assays are provided for identifying anti-TfR antibodiesthereof having biological activity. Biological activity may include,e.g., transporting a compound associated with/conjugated to the antibodyacross the BBB into the brain and/or CNS. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-TfRantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In some embodiments, the anti-TfR antibody herein is coupled with aneurological disorder drug, a chemotherapeutic agent and/or an imagingagent in order to more efficiently transport the drug, chemotherapeuticagent and/or the imaging agent across the BBB.

Covalent conjugation can either be direct or via a linker. In certainembodiments, direct conjugation is by construction of a protein fusion(i.e., by genetic fusion of the two genes encoding the anti-TfR antibodyand e.g., the neurological disorder drug and expression as a singleprotein). In certain embodiments, direct conjugation is by formation ofa covalent bond between a reactive group on one of the two portions ofthe anti-TfR antibody and a corresponding group or acceptor on the,e.g., neurological drug. In certain embodiments, direct conjugation isby modification (i.e., genetic modification) of one of the two moleculesto be conjugated to include a reactive group (as nonlimiting examples, asulfhydryl group or a carboxyl group) that forms a covalent attachmentto the other molecule to be conjugated under appropriate conditions. Asone nonlimiting example, a molecule (i.e., an amino acid) with a desiredreactive group (i.e., a cysteine residue) may be introduced into theanti-TfR antibody and a disulfide bond formed with the e.g.,neurological drug. Methods for covalent conjugation of nucleic acids toproteins are also known in the art (i.e., photocrosslinking, see, e.g.,Zatsepin et al. Russ. Chem. Rev. 74: 77-95 (2005))

Non-covalent conjugation can be by any nonconvalent attachment means,including hydrophobic bonds, ionic bonds, electrostatic interactions,and the like, as will be readily understood by one of ordinary skill inthe art.

Conjugation may also be performed using a variety of linkers. Forexample, an anti-TfR antibody and a neurological drug may be conjugatedusing a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Peptidelinkers, comprised of from one to twenty amino acids joined by peptidebonds, may also be used. In certain such embodiments, the amino acidsare selected from the twenty naturally-occurring amino acids. In certainother such embodiments, one or more of the amino acids are selected fromglycine, alanine, proline, asparagine, glutamine and lysine. The linkermay be a “cleavable linker” facilitating release of the neurologicaldrug upon delivery to the brain. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The invention herein expressly contemplates, but is not limited to,conjugates prepared with cross-linker reagents including, but notlimited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

In some embodiments, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-TfR antibodies provided hereinis useful for detecting the presence of TfR in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue, such as blood (i.e., immature red blood cells), CSF, andBBB-containing tissue.

In some embodiments, an anti-TfR antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of TfR in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-TfR antibody as described herein under conditionspermissive for binding of the anti-TfR antibody to TfR, and detectingwhether a complex is formed between the anti-TfR antibody and TfR. Suchmethod may be an in vitro or in vivo method. In some embodiments, ananti-TfR antibody is used to select subjects eligible for therapy withan anti-TfR antibody, e.g. where TfR is a biomarker for selection ofpatients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include disorders involving immature red blood cells, due tothe fact that TfR is expressed in reticulocytes and is thereforedetectable by any of the antibodies of the invention. Such disordersinclude anemia and other disorders arising from reduced levels ofreticulocytes, or congenital polycythemia or neoplastic polycythemiavera, where raised red blood cell counts due to hyperproliferation of,e.g., reticulocytes, results in thickening of blood and concomitantphysiological symptoms.

In certain embodiments, labeled anti-TfR antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In some embodiments, the intact antibody lacks effector function. Inanother embodiment, the intact antibody has reduced effector function.In another embodiment, the intact antibody is engineered to have reducedeffector function. In some aspects, the antibody is a Fab. In anotheraspect, the antibody has one or more Fc mutations reducing oreliminating effector function. In another aspect, the antibody hasmodified glycosylation due, e.g., to producing the antibody in a systemlacking normal human glycosylation enzymes. In another aspect, the Igbackbone is modified to one which naturally possesses limited or noeffector function.

Various techniques are available for determining binding of the antibodyto the TfR. One such assay is an enzyme linked immunosorbent assay(ELISA) for confirming an ability to bind to human TfR (and brainantigen). According to this assay, plates coated with antigen (e.g.recombinant TfR) are incubated with a sample comprising the anti-TfRantibody and binding of the antibody to the antigen of interest isdetermined.

Assays for evaluating uptake of systemically administered antibody andother biological activity of the antibody can be performed as disclosedin the examples or as known for the anti-CNS antigen antibody ofinterest.

In some aspects, assays are provided for identifying anti-TfR antibodiesconjugated (either covalently or non-covalently) to anti-BACE1antibodies having biological activity. Biological activity may include,e.g., inhibition of BACE1 aspartyl protease activity. Antibodies havingsuch biological activity in vivo and/or in vitro are also provided, e.g.as evaluated by homogeneous time-resolved fluorescence HTRF assay or amicrofluidic capillary electrophoretic (MCE) assay using syntheticsubstrate peptides, or in vivo in cell lines which express BACE1substrates such as APP.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-TfR as described herein areprepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of lyophilized formulationsor aqueous solutions. Pharmaceutically acceptable carriers, excipients,or stabilizers are generally nontoxic to recipients at the dosages andconcentrations employed, and include, but are not limited to: bufferssuch as phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In some aspects, a sHASEGP is combinedwith one or more additional glycosaminoglycanases such aschondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide one or moreactive ingredients for treating a neuropathy disorder, aneurodegenerative disease, cancer, an ocular disease disorder, a seizuredisorder, a lysosomal storage disease, an amyloidosis, a viral ormicrobial disease, ischemia, a behavioral disorder or CNS inflammation.Exemplary such medicaments are discussed hereinbelow. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in, for example, Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). One or moreactive ingredients may be encapsulated in liposomes that are coupled toanti-TfR antibodies described herein (see e.g., U.S. Patent ApplicationPublication No. 20020025313).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Nonlimitingexamples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-TfR antibodies provided herein may be used intherapeutic methods. In some aspects, an anti-TfR antibody for use as amedicament is provided. For example, the invention provides a method oftransporting a therapeutic compound across the blood-brain barrier withreduced or eliminated impact on red blood cell populations comprisingexposing the anti-TfR antibody coupled to a therapeutic compound (e.g. amultispecific antibody which binds both the TfR and a brain antigen) tothe BBB such that the antibody transports the therapeutic compoundcoupled thereto across the BBB. In another example, the inventionprovides a method of transporting a neurological disorder drug acrossthe blood-brain barrier comprising exposing an anti-TfR antibody of theinvention coupled to a brain disorder drug (e.g. a multispecificantibody which binds both the TfR and a brain antigen) to the BBB suchthat the antibody transports the neurological disorder drug coupledthereto across the BBB with reduced or eliminated impact on red bloodcell populations. In some embodiments, the BBB is in a mammal (e.g. ahuman), e.g. one which has a neurological disorder, including, withoutlimitation: Alzheimer's disease (AD), stroke, dementia, musculardystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis(ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome,Parkinson's disease, Pick's disease, Paget's disease, cancer, traumaticbrain injury, etc.

In some embodiments, the neurological disorder is selected from: aneuropathy, an amyloidosis, cancer (e.g. involving the CNS or brain), anocular disease or disorder, a viral or microbial infection, inflammation(e.g. of the CNS or brain), ischemia, neurodegenerative disease,seizure, behavioral disorder, lysosomal storage disease, etc. Theantibodies of the invention are particularly suited to treatment of suchneurological disorders due to their ability to transport one or moreassociated active ingredients/coupled therapeutic compounds across theBBB and into the CNS/brain where such disorders find their molecular,cellular, or viral/microbial basis.

Neuropathy disorders are diseases or abnormalities of the nervous systemcharacterized by inappropriate or uncontrolled nerve signaling or lackthereof, and include, but are not limited to, chronic pain (includingnociceptive pain), pain caused by an injury to body tissues, includingcancer-related pain, neuropathic pain (pain caused by abnormalities inthe nerves, spinal cord, or brain), and psychogenic pain (entirely ormostly related to a psychological disorder), headache, migraine,neuropathy, and symptoms and syndromes often accompanying suchneuropathy disorders such as vertigo or nausea.

For a neuropathy disorder, a neurological drug may be selected that isan analgesic including, but not limited to, a narcotic/opioid analgesic(i.e., morphine, fentanyl, hydrocodone, meperidine, methadone,oxymorphone, pentazocine, propoxyphene, tramadol, codeine andoxycodone), a nonsteroidal anti-inflammatory drug (NSAID) (i.e.,ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam,nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin), acorticosteroid (i.e., cortisone, prednisone, prednisolone,dexamethasone, methylprednisolone and triamcinolone), an anti-migraineagent (i.e., sumatriptin, almotriptan, frovatriptan, sumatriptan,rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan andergotamine), acetaminophen, a salicylate (i.e., aspirin, cholinesalicylate, magnesium salicylate, diflunisal, and salsalate), aanti-convulsant (i.e., carbamazepine, clonazepam, gabapentin,lamotrigine, pregabalin, tiagabine, and topiramate), an anaesthetic(i.e., isoflurane, trichloroethylene, halothane, sevoflurane,benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine,propoxycaine, procaine, novocaine, proparacaine, tetracaine, articaine,bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine,lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine,trimecaine, saxitoxin and tetrodotoxin), and a cox-2-inhibitor (i.e.,celecoxib, rofecoxib, and valdecoxib). For a neuropathy disorder withvertigo involvement, a neurological drug may be selected that is ananti-vertigo agent including, but not limited to, meclizine,diphenhydramine, promethazine and diazepam. For a neuropathy disorderwith nausea involvement, a neurological drug may be selected that is ananti-nausea agent including, but not limited to, promethazine,chlorpromazine, prochlorperazine, trimethobenzamide, and metoclopramide.

Amyloidoses are a group of diseases and disorders associated withextracellular proteinaceous deposits in the CNS, including, but notlimited to, secondary amyloidosis, age-related amyloidosis, Alzheimer'sDisease (AD), mild cognitive impairment (MCI), Lewy body dementia,Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutchtype); the Guam Parkinson-Dementia complex, cerebral amyloid angiopathy,Huntington's disease, progressive supranuclear palsy, multiplesclerosis; Creutzfeld Jacob disease, Parkinson's disease, transmissiblespongiform encephalopathy, HIV-related dementia, amyotropic lateralsclerosis (ALS), inclusion-body myositis (IBM), and ocular diseasesrelating to beta-amyloid deposition (i.e., macular degeneration,drusen-related optic neuropathy, and cataract).

For amyloidosis, a neurological drug may be selected that includes, butis not limited to, an antibody or other binding molecule (including, butnot limited to a small molecule, a peptide, an aptamer, or other proteinbinder) that specifically binds to a target selected from: betasecretase, tau, presenilin, amyloid precursor protein or portionsthereof, amyloid beta peptide or oligomers or fibrils thereof, deathreceptor 6 (DR6), receptor for advanced glycation endproducts (RAGE),parkin, and huntingtin; a cholinesterase inhibitor (i.e., galantamine,donepezil, rivastigmine and tacrine); an NMDA receptor antagonist (i.e.,memantine), a monoamine depletor (i.e., tetrabenazine); an ergoloidmesylate; an anticholinergic antiparkinsonism agent (i.e., procyclidine,diphenhydramine, trihexylphenidyl, benztropine, biperiden andtrihexyphenidyl); a dopaminergic antiparkinsonism agent (i.e.,entacapone, selegiline, pramipexole, bromocriptine, rotigotine,selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa,pergolide, tolcapone and amantadine); a tetrabenazine; ananti-inflammatory (including, but not limited to, a nonsteroidalanti-inflammatory drug (i.e., indomethicin and other compounds listedabove); a hormone (i.e., estrogen, progesterone and leuprolide); avitamin (i.e., folate and nicotinamide); a dimebolin; a homotaurine(i.e., 3-aminopropanesulfonic acid; 3APS); a serotonin receptor activitymodulator (i.e., xaliproden); an, an interferon, and a glucocorticoid.

Cancers of the CNS are characterized by aberrant proliferation of one ormore CNS cell (i.e., a neural cell) and include, but are not limited to,glioma, glioblastoma multiforme, meningioma, astrocytoma, acousticneuroma, chondroma, oligodendroglioma, medulloblastomas, ganglioglioma,Schwannoma, neurofibroma, neuroblastoma, and extradural, intramedullaryor intradural tumors.

For cancer, a neurological drug may be selected that is achemotherapeutic agent. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphor-amide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition of chemotherapeutic agents areanti-hormonal agents that act to regulate, reduce, block, or inhibit theeffects of hormones that can promote the growth of cancer, and are oftenin the form of systemic, or whole-body treatment. They may be hormonesthemselves. Examples include anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; anti-progesterones; estrogen receptordown-regulators (ERDs); agents that function to suppress or shut downthe ovaries, for example, leutinizing hormone-releasing hormone (LHRH)agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelinacetate, buserelin acetate and tripterelin; other anti-androgens such asflutamide, nilutamide and bicalutamide; and aromatase inhibitors thatinhibit the enzyme aromatase, which regulates estrogen production in theadrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, andARIMIDEX® anastrozole. In addition, such definition of chemotherapeuticagents includes bisphosphonates such as clodronate (for example,BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® Rzoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate,SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine(a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH;lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinasesmall-molecule inhibitor also known as GW572016); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Another group of compounds that may be selected as neurological drugsfor cancer treatment or prevention are anti-cancer immunoglobulins(including, but not limited to, trastuzumab, pertuzumab, bevacizumab,alemtuxumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan,panitumumab and rituximab). In some instances, antibodies in conjunctionwith a toxic label or conjugate may be used to target and kill desiredcells (i.e., cancer cells), including, but not limited to, tositumomabwith a ¹³¹I radiolabel, or trastuzumab emtansine.

Ocular diseases or disorders are diseases or disorders of the eye, whichfor the purposes herein is considered a CNS organ segregated by the BBB.Ocular diseases or disorders include, but are not limited to, disordersof sclera, cornea, iris and ciliary body (i.e., scleritis, keratitis,corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson'ssuperficial punctate keratopathy, corneal neovascularisation, Fuchs'dystrophy, keratoconus, keratoconjunctivitis sicca, iritis and uveitis),disorders of the lens (i.e., cataract), disorders of choroid and retina(i.e., retinal detachment, retinoschisis, hypertensive retinopathy,diabetic retinopathy, retinopathy, retinopathy of prematurity,age-related macular degeneration, macular degeneration (wet or dry),epiretinal membrane, retinitis pigmentosa and macular edema), glaucoma,floaters, disorders of optic nerve and visual pathways (i.e., Leber'shereditary optic neuropathy and optic disc drusen), disorders of ocularmuscles/binocular movement accommodation/refraction (i.e., strabismus,ophthalmoparesis, progressive external opthalmoplegia, esotropia,exotropia, hypermetropia, myopia, astigmatism, anisometropia, presbyopiaand ophthalmoplegia), visual disturbances and blindness (i.e.,amblyopia, Lever's congenital amaurosis, scotoma, color blindness,achromatopsia, nyctalopia, blindness, river blindness andmicro-opthalmia/coloboma), red eye, Argyll Robertson pupil,keratomycosis, xerophthalmia and andaniridia.

For an ocular disease or disorder, a neurological drug may be selectedthat is an anti-angiogenic ophthalmic agent (i.e., bevacizumab,ranibizumab and pegaptanib), an ophthalmic glaucoma agent (i.e.,carbachol, epinephrine, demecarium bromide, apraclonidine, brimonidine,brinzolamide, levobunolol, timolol, betaxolol, dorzolamide, bimatoprost,carteolol, metipranolol, dipivefrin, travoprost and latanoprost), acarbonic anhydrase inhibitor (i.e., methazolamide and acetazolamide), anophthalmic antihistamine (i.e., naphazoline, phenylephrine andtetrahydrozoline), an ocular lubricant, an ophthalmic steroid (i.e.,fluorometholone, prednisolone, loteprednol, dexamethasone,difluprednate, rimexolone, fluocinolone, medrysone and triamcinolone),an ophthalmic anesthetic (i.e., lidocaine, proparacaine and tetracaine),an ophthalmic anti-infective (i.e., levofloxacin, gatifloxacin,ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin/polymyxin b,sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin,sulfisoxazole, gentamicin, idoxuridine, erythromycin, natamycin,gramicidin, neomycin, ofloxacin, trifluridine, ganciclovir, vidarabine),an ophthalmic anti-inflammatory agent (i.e., nepafenac, ketorolac,flurbiprofen, suprofen, cyclosporine, triamcinolone, diclofenac andbromfenac), and an ophthalmic antihistamine or decongestant (i.e.,ketotifen, olopatadine, epinastine, naphazoline, cromolyn,tetrahydrozoline, pemirolast, bepotastine, naphazoline, phenylephrine,nedocromil, lodoxamide, phenylephrine, emedastine and azelastine).

Viral or microbial infections of the CNS include, but are not limitedto, infections by viruses (i.e., influenza, HIV, poliovirus, rubella),bacteria (i.e., Neisseria sp., Streptococcus sp., Pseudomonas sp.,Proteus sp., E. coli, S. aureus, Pneumococcus sp., Meningococcus sp.,Haemophilus sp., and Mycobacterium tuberculosis) and othermicroorganisms such as fungi (i.e., yeast, Cryptococcus neoformans),parasites (i.e., Toxoplasma gondii) or amoebas resulting in CNSpathophysiologies including, but not limited to, meningitis,encephalitis, myelitis, vasculitis and abscess, which can be acute orchronic.

For a viral or microbial disease, a neurological drug may be selectedthat includes, but is not limited to, an antiviral compound (including,but not limited to, an adamantane antiviral (i.e., rimantadine andamantadine), an antiviral interferon (i.e., peginterferon alfa-2b), achemokine receptor antagonist (i.e., maraviroc), an integrase strandtransfer inhibitor (i.e., raltegravir), a neuraminidase inhibitor (i.e.,oseltamivir and zanamivir), a non-nucleoside reverse transcriptaseinhibitor (i.e., efavirenz, etravirine, delavirdine and nevirapine), anucleoside reverse transcriptase inhibitors (tenofovir, abacavir,lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir,zalcitabine, telbivudine and didanosine), a protease inhibitor (i.e.,darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir,amprenavir, indinavir and saquinavir), a purine nucleoside (i.e.,valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir,valganciclovir and cidofovir), and a miscellaneous antiviral (i.e.,enfuvirtide, foscarnet, palivizumab and fomivirsen)), an antibiotic(including, but not limited to, an aminopenicillin (i.e., amoxicillin,ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin,flucoxacillin, temocillin, azlocillin, carbenicillin, ticarcillin,mezlocillin, piperacillin and bacampicillin), a cephalosporin (i.e.,cefazolin, cephalexin, cephalothin, cefamandole, ceftriaxone,cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine,loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin),a carbapenemipenem (i.e., imipenem, meropenem, ertapenem, faropenem anddoripenem), a monobactam (i.e., aztreonam, tigemonam, norcardicin A andtabtoxinine-beta-lactam, a beta-lactamase inhibitor (i.e., clavulanicacid, tazobactam and sulbactam) in conjunction with another beta-lactamantibiotic, an aminoglycoside (i.e., amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), anansamycin (i.e., geldanamycin and herbimycin), a carbacephem (i.e.,loracarbef), a glycopeptides (i.e., teicoplanin and vancomycin), amacrolide (i.e., azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin andspectinomycin), a monobactam (i.e., aztreonam), a quinolone (i.e.,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin and temafloxacin), a sulfonamide (i.e., mafenide,sulfonamidochrysoidine, sulfacetamide, sulfadiazine, sulfamethizole,sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprimand sulfamethoxazole), a tetracycline (i.e., tetracycline,demeclocycline, doxycycline, minocycline and oxytetracycline), anantineoplastic or cytotoxic antibiotic (i.e., doxorubicin, mitoxantrone,bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin,plicamycin, mitomycin, pentostatin and valrubicin) and a miscellaneousantibacterial compound (i.e., bacitracin, colistin and polymyxin B)), anantifungal (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine,iodoquinol and paromomycin), and an antiparasitic (including, but notlimited to, quinine, chloroquine, amodiaquine, pyrimethamine,sulphadoxine, proguanil, mefloquine, atovaquone, primaquine,artemesinin, halofantrine, doxycycline, clindamycin, mebendazole,pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin,rifampin, amphotericin B, melarsoprol, efornithine and albendazole).

Inflammation of the CNS includes, but is not limited to, inflammationthat is caused by an injury to the CNS, which can be a physical injury(i.e., due to accident, surgery, brain trauma, spinal cord injury,concussion) and an injury due to or related to one or more otherdiseases or disorders of the CNS (i.e., abscess, cancer, viral ormicrobial infection).

For CNS inflammation, a neurological drug may be selected that addressesthe inflammation itself (i.e., a nonsteroidal anti-inflammatory agentsuch as ibuprofen or naproxen), or one which treats the underlying causeof the inflammation (i.e., an anti-viral or anti-cancer agent).

Ischemia of the CNS, as used herein, refers to a group of disordersrelating to aberrant blood flow or vascular behavior in the brain or thecauses therefor, and includes, but is not limited to: focal brainischemia, global brain ischemia, stroke (i.e., subarachnoid hemorrhageand intracerebral hemorrhage), and aneurysm.

For ischemia, a neurological drug may be selected that includes, but isnot limited to, a thrombolytic (i.e., urokinase, alteplase, reteplaseand tenecteplase), a platelet aggregation inhibitor (i.e., aspirin,cilostazol, clopidogrel, prasugrel and dipyridamole), a statin (i.e.,lovastatin, pravastatin, fluvastatin, rosuvastatin, atorvastatin,simvastatin, cerivastatin and pitavastatin), and a compound to improveblood flow or vascular flexibility, including, e.g., blood pressuremedications.

Neurodegenerative diseases are a group of diseases and disordersassociated with neural cell loss of function or death in the CNS, andinclude, but are not limited to: adrenoleukodystrophy, Alexander'sdisease, Alper's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease, cockayne syndrome, corticobasaldegeneration, degeneration caused by or associated with an amyloidosis,Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy'sdisease, multiple system atrophy, multiple sclerosis, primary lateralsclerosis, progressive supranuclear palsy, spinal muscular atrophy,transverse myelitis, Refsum's disease, and spinocerebellar ataxia.

For a neurodegenerative disease, a neurological drug may be selectedthat is a growth hormone or neurotrophic factor; examples include butare not limited to brain-derived neurotrophic factor (BDNF), nervegrowth factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocytegrowth factor (HGF), epidermal growth factor (EGF), transforming growthfactor (TGF)-alpha, TGF-beta, vascular endothelial growth factor (VEGF),interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor(CNTF), glial-derived neurotrophic factor (GDNF), neurturin,platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin,persephin, interleukins, glial cell line derived neurotrophic factor(GFR), granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, and stemcell factor (SCF).

Seizure diseases and disorders of the CNS involve inappropriate and/orabnormal electrical conduction in the CNS, and include, but are notlimited to epilepsy (i.e., absence seizures, atonic seizures, benignRolandic epilepsy, childhood absence, clonic seizures, complex partialseizures, frontal lobe epilepsy, febrile seizures, infantile spasms,juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastautsyndrome, Landau-Kleffner Syndrome, Dravet's syndrome, Otahara syndrome,West syndrome, myoclonic seizures, mitochondrial disorders, progressivemyoclonic epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen'sSyndrome, simple partial seizures, secondarily generalized seizures,temporal lobe epilepsy, toniclonic seizures, tonic seizures, psychomotorseizures, limbic epilepsy, partial-onset seizures, generalized-onsetseizures, status epilepticus, abdominal epilepsy, akinetic seizures,autonomic seizures, massive bilateral myoclonus, catamenial epilepsy,drop seizures, emotional seizures, focal seizures, gelastic seizures,Jacksonian March, Lafora Disease, motor seizures, multifocal seizures,nocturnal seizures, photosensitive seizure, pseudo seizures, sensoryseizures, subtle seizures, sylvan seizures, withdrawal seizures, andvisual reflex seizures).

For a seizure disorder, a neurological drug may be selected that is ananticonvulsant or antiepileptic including, but not limited to,barbiturate anticonvulsants (i.e., primidone, metharbital,mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal,barbital, brallobarbital and phenobarbital), benzodiazepineanticonvulsants (i.e., diazepam, clonazepam, and lorazepam), carbamateanticonvulsants (i.e. felbamate), carbonic anhydrase inhibitoranticonvulsants (i.e., acetazolamide, topiramate and zonisamide),dibenzazepine anticonvulsants (i.e., rufinamide, carbamazepine, andoxcarbazepine), fatty acid derivative anticonvulsants (i.e., divalproexand valproic acid), gamma-aminobutyric acid analogs (i.e., pregabalin,gabapentin and vigabatrin), gamma-aminobutyric acid reuptake inhibitors(i.e., tiagabine), gamma-aminobutyric acid transaminase inhibitors(i.e., vigabatrin), hydantoin anticonvulsants (i.e. phenytoin, ethotoin,fosphenytoin and mephenytoin), miscellaneous anticonvulsants (i.e.,lacosamide and magnesium sulfate), progestins (i.e., progesterone),oxazolidinedione anticonvulsants (i.e., paramethadione andtrimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam),succinimide anticonvulsants (i.e., ethosuximide and methsuximide),triazine anticonvulsants (i.e., lamotrigine), and urea anticonvulsants(i.e., phenacemide and pheneturide).

Behavioral disorders are disorders of the CNS characterized by aberrantbehavior on the part of the afflicted subject and include, but are notlimited to: sleep disorders (i.e., insomnia, parasomnias, night terrors,circadian rhythm sleep disorders, and narcolepsy), mood disorders (i.e.,depression, suicidal depression, anxiety, chronic affective disorders,phobias, panic attacks, obsessive-compulsive disorder, attention deficithyperactivity disorder (ADHD), attention deficit disorder (ADD), chronicfatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolardisorder), eating disorders (i.e., anorexia or bulimia), psychoses,developmental behavioral disorders (i.e., autism, Rett's syndrome,Aspberger's syndrome), personality disorders and psychotic disorders(i.e., schizophrenia, delusional disorder, and the like).

For a behavioral disorder, a neurological drug may be selected from abehavior-modifying compound including, but not limited to, an atypicalantipsychotic (i.e., risperidone, olanzapine, apripiprazole, quetiapine,paliperidone, asenapine, clozapine, iloperidone and ziprasidone), aphenothiazine antipsychotic (i.e., prochlorperazine, chlorpromazine,fluphenazine, perphenazine, trifluoperazine, thioridazine andmesoridazine), a thioxanthene (i.e., thiothixene), a miscellaneousantipsychotic (i.e., pimozide, lithium, molindone, haloperidol andloxapine), a selective serotonin reuptake inhibitor (i.e., citalopram,escitalopram, paroxetine, fluoxetine and sertraline), aserotonin-norepinephrine reuptake inhibitor (i.e., duloxetine,venlafaxine, desvenlafaxine, a tricyclic antidepressant (i.e., doxepin,clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine,imipramine, protriptyline and desipramine), a tetracyclic antidepressant(i.e., mirtazapine and maprotiline), a phenylpiperazine antidepressant(i.e., trazodone and nefazodone), a monoamine oxidase inhibitor (i.e.,isocarboxazid, phenelzine, selegiline and tranylcypromine), abenzodiazepine (i.e., alprazolam, estazolam, flurazeptam, clonazepam,lorazepam and diazepam), a norepinephrine-dopamine reuptake inhibitor(i.e., bupropion), a CNS stimulant (i.e., phentermine, diethylpropion,methamphetamine, dextroamphetamine, amphetamine, methylphenidate,dexmethylphenidate, lisdexamfetamine, modafinil, pemoline,phendimetrazine, benzphetamine, phendimetrazine, armodafinil,diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), ananxiolytic/sedative/hypnotic (including, but not limited to, abarbiturate (i.e., secobarbital, phenobarbital and mephobarbital), abenzodiazepine (as described above), and a miscellaneousanxiolytic/sedative/hypnotic (i.e. diphenhydramine, sodium oxybate,zaleplon, hydroxyzine, chloral hydrate, aolpidem, buspirone, doxepin,eszopiclone, ramelteon, meprobamate and ethclorvynol)), a secretin (see,e.g., Ratliff-Schaub et al. Autism 9: 256-265 (2005)), an opioid peptide(see, e.g., Cowen et al., J. Neurochem. 89:273-285 (2004)), and aneuropeptide (see, e.g., Hethwa et al. Am. J. Physiol. 289: E301-305(2005)).

Lysosomal storage disorders are metabolic disorders which are in somecases associated with the CNS or have CNS-specific symptoms; suchdisorders include, but are not limited to: Tay-Sachs disease, Gaucher'sdisease, Fabry disease, mucopolysaccharidosis (types I, II, III, IV, V,VI and VII), glycogen storage disease, GM1-gangliosidosis, metachromaticleukodystrophy, Farber's disease, Canavan's leukodystrophy, and neuronalceroid lipofuscinoses types 1 and 2, Niemann-Pick disease, Pompedisease, and Krabbe's disease.

For a lysosomal storage disease, a neurological drug may be selectedthat is itself or otherwise mimics the activity of the enzyme that isimpaired in the disease. Exemplary recombinant enzymes for the treatmentof lysosomal storage disorders include, but are not limited to those setforth in e.g., U.S. Patent Application publication no. 2005/0142141(i.e., alpha-L-iduronidase, iduronate-2-sulphatase, N-sulfatase,alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase,beta-galactosidase, arylsulphatase B, beta-glucuronidase, acidalpha-glucosidase, glucocerebrosidase, alpha-galactosidase A,hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase,beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase,palmitoyl-protein thioesterase 1 and tripeptidyl amino peptidase 1).

In another embodiment, diseases related to or caused by inappropriateoverproduction of red blood cells, or wherein the overproduction of redblood cells is an effect of the disease, can be prevented or treated bythe reticulocyte-depleting effect recognized herein of anti-TfRantibodies retaining at least partial effector function. For example, incongenital or neoplastic polycythemia vera, elevated red blood cellcounts due to hyperproliferation of, e.g., reticulocytes, results inthickening of blood and concomitant physiological symptoms (d'Onofrio etal., Clin. Lab. Haematol. (1996) Suppl. 1: 29-34). Administration of ananti-TfR antibody of the invention wherein at least partial effectorfunction of the antibody was preserved would permit selective removal ofimmature reticulocyte populations without impacting normal transferrintransport into the CNS. Dosing of such an antibody could be modulatedsuch that acute clinical symptoms could be minimized (ie, by dosing at avery low dose or at widely-spaced intervals), as well-understood in theart.

In some aspects, an antibody of the invention is used to detect aneurological disorder before the onset of symptoms and/or to assess theseverity or duration of the disease or disorder. In some aspects, theantibody permits detection and/or imaging of the neurological disorder,including imaging by radiography, tomography, or magnetic resonanceimaging (MRI).

In some aspects, a low affinity anti-TfR antibody of the invention foruse as a medicament is provided. In further aspects, a low affinityanti-TfR antibody for use in treating a neurological disease or disorder(e.g., Alzheimer's disease) without depleting red blood cells (ie,reticulocytes) is provided. In certain embodiments, a modified lowaffinity anti-TfR antibody for use in a method of treatment as describedherein is provided. In certain embodiments, the invention provides a lowaffinity anti-TfR antibody modified to improve its safety for use in amethod of treating an individual having a neurological disease ordisorder comprising administering to the individual an effective amountof the anti-TfR antibody (optionally coupled to a neurological disorderdrug). In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent. In further embodiments, the inventionprovides an anti-TfR antibody modified to improve its safety for use inreducing or inhibiting amlyoid plaque formation in a patient at risk orsuffering from a neurological disease or disorder (e.g., Alzheimer'sdisease). An “individual” according to any of the above embodiments isoptionally a human. In certain aspects, the anti-TfR antibody of theinvention for use in the methods of the invention improves uptake of theneurological disorder drug with which it is coupled.

In a further aspect, the invention provides for the use of a lowaffinity anti-TfR antibody of the invention in the manufacture orpreparation of a medicament. In some embodiments, the medicament is fortreatment of neurological disease or disorder. In a further embodiment,the medicament is for use in a method of treating neurological diseaseor disorder comprising administering to an individual havingneurological disease or disorder an effective amount of the medicament.In one such embodiment, the method further comprises administering tothe individual an effective amount of at least one additionaltherapeutic agent.

In a further aspect, the invention provides a method for treatingAlzheimer's disease. In some embodiments, the method comprisesadministering to an individual having Alzheimer's disease an effectiveamount of a multispecific antibody of the invention which binds bothBACE1 and TfR or both Abeta and TfR. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent. An “individual” according toany of the above embodiments may be a human.

The anti-TfR antibodies of the invention can be used either alone or incombination with other agents in a therapy. For instance, the anti-TfRantibody of the invention may be co-administered with at least oneadditional therapeutic agent. In certain embodiments, an additionaltherapeutic agent is a therapeutic agent effective to treat the same ora different neurological disorder as the anti-TfR antibody is beingemployed to treat. Exemplary additional therapeutic agents include, butare not limited to: the various neurological drugs described above,cholinesterase inhibitors (such as donepezil, galantamine, rovastigmine,and tacrine), NMDA receptor antagonists (such as memantine), amyloidbeta peptide aggregation inhibitors, antioxidants, γ-secretasemodulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARγagonists, HMS-CoA reductase inhibitors (statins), ampakines, calciumchannel blockers, GABA receptor antagonists, glycogen synthase kinaseinhibitors, intravenous immunoglobulin, muscarinic receptor agonists,nicrotinic receptor modulators, active or passive amyloid beta peptideimmunization, phosphodiesterase inhibitors, serotonin receptorantagonists and anti-amyloid beta peptide antibodies. In certainembodiments, the at least one additional therapeutic agent is selectedfor its ability to mitigate one or more side effects of the neurologicaldrug.

As exemplified herein, certain anti-TfR antibodies may have side effectsthat negatively impact reticulocyte populations in a subject treatedwith the anti-TfR antibody. Thus, in certain embodiments, at least onefurther therapeutic agent selected for its ability to mitigate suchnegative side effect on reticulocyte populations is coadministered withan anti-TfR antibody of the invention. Examples of such therapeuticagents include, but are not limited to, agents to increase red bloodcell (ie, reticulocyte) populations, agents to support growth anddevelopment of red blood cells (ie, reticulocytes), and agents toprotect red blood cell populations from the effects of the anti-TfRantibody; such agents include, but are not limited to, erythropoietin(EPO), iron supplements, vitamin C, folic acid, and vitamin B12, as wellas physical replacement of red blood cells (ie, reticulocytes) by, forexample, transfusion with similar cells, which may be from anotherindividual of similar blood type or may have been previously extractedfrom the subject to whom the anti-TfR antibody is administered. It willbe understood by one of ordinary skill in the art that in someinstances, agents intended to protect existing red blood cells (ie,reticulocytes) are preferably administered to the subject preceding orconcurrent with the anti-TfR antibody therapy, while agents intended tosupport or initiate the regrowth/development of red blood cells or bloodcell populations (ie, reticulocytes or reticulocyte populations) arepreferably administered concurrent with or after the anti-TfR antibodytherapy such that such blood cells can be replenished after the anti-TfRantibody treatment.

In certain other such embodiments, the at least one further therapeuticagent is selected for its ability to inhibit or prevent the activationof the complement pathway upon administration of the anti-TfR antibody.Examples of such therapeutic agents include, but are not limited to,agents that interfere with the ability of the anti-TfR antibody to bindto or activate the complement pathway and agents that inhibit one ormore molecular interactions within the complement pathway, and aredescribed generally in Mollnes and Kirschfink (2006) Molec. Immunol43:107-121, the contents of which are expressly incorporated herein byreference.

Such combination therapies noted above and herein encompass combinedadministration (where two or more therapeutic agents are included in thesame or separate formulations), and separate administration, in whichcase, administration of the antibody of the invention can occur priorto, simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. In some embodiments, administrationof the anti-TfR antibody and administration of an additional therapeuticagent occur within about one month, or within about one, two or threeweeks, or within about one, two, three, four, five or six days, of eachother. Antibodies of the invention can also be used in combination withother interventional therapies such as, but not limited to, radiationtherapy, behavioral therapy, or other therapies known in the art andappropriate for the neurological disorder to be treated or prevented.

An anti-TfR antibody of the invention (and any additional therapeuticagent) can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question or toprevent, mitigate or ameliorate one or more side effects of antibodyadministration. The effective amount of such other agents depends on theamount of antibody present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 40 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg, 5.0 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg,30 mg/kg, 35 mg/kg or 40 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. However, other dosage regimens maybe useful. It will be appreciated that one method to reduce impact onreticulocyte populations by administration of anti-TfR antibodies is tomodify the amount or timing of the doses such that overall lowerquantities of circulating antibody are present in the bloodstream tointeract with reticulocytes. In one nonlimiting example, a lower dose ofthe anti-TfR antibodies may be administered with greater frequency thana higher dose would be. The dosage used may be balanced between theamount of antibody necessary to be delivered to the CNS (itself relatedto the affinity of the CNS antigen-specific portion of the antibody),the affinity of that antibody for TfR, and whether or not red blood cell(ie, reticulocyte)-protecting, growth and development-stimulating, orcomplement pathway-inhibiting compound(s) are being co- or seriallyadministered with the antibody. The progress of this therapy is easilymonitored by conventional techniques and assays as described herein andas known in the art.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-TfR antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-TfR antibody.

EXAMPLES Example 1: Generation, Characterization and Humanization ofHuman/Cyno Cross-Reactive Anti-TfR Antibodies

Initially, a naïve antibody phage panning process was performed in anattempt to identify antibodies cross-reactive with both human TfR andTfR from cynomolgous (“cyno”) monkeys that further did not compete withTf for binding to TfR (Lee et al. JMB (2004) 1073-1093). No suchcross-reactive, non-Tf-competing clone was identified from this phagepanning process. However, two antibodies were identified that wereuseful in characterizing subsequently generated hybridoma clones.

A species cross-reactive antibody was identified that competes with Tffor binding to human or cyno TfR (Tf-competing antibody). The epitope ofanother clone, specific for human TfR, was mapped to the apical domainof huTfR using mouse/human chimeric TfR receptors (FIG. 1). This apicaldomain-binding clone lost binding to huTfR when the mouse TfR sequencein the apical domain was substituted into huTfR.

Next, an immunization-based approach to generate cross-reactiveanti-human/cyno TfR antibodies was performed. Human TfR extracellulardomain (“ecd”) containing an N-terminal His tag and humanhemachromatosis protein (“HFE”) were expressed and purified as described(Bennet et al, Nature (2000) 403, 46-53). An analogous cyno TfR ecdconstruct was also made. Cyno TfR was expressed and purified in asimilar manner. Human and cyno cross-reactive TfR antibodies weregenerated by immunizing 5 Balb/C mice in the footpad with 6 doses (twiceper week) containing 2 μg each of cynoTfR and huTfR ecd. All mice serawere FACS positive and all mice were fused. Of 1632 hybridomas screened,111 were ELISA positive for binding to both human and cyno TfR.

The resulting ELISA-positive hybridomas were screened by FACS in thepresence of 1 μM human holo-Tf for binding to 293 cells transientlyexpressing human or cyno TfR. Briefly, FACS analysis was performed using293 cells transfected with full length human or cyno TfR usinglipofectamin 2000 plus (Invitrogen) 48-72 h before FACS analysis.Non-transfected (control) and transfected 293 cells were washed twicewith FACS buffer (PBS containing 1% BSA), 50 μM of hybridoma supernatant(normalized to 10 μg/ml) was added to 293 cells in the presence of 1 μMhuman holo-Tf and incubated on ice for 30 min. Cells were washed twicewith FACS buffer, 50 μl of PE-Goat-anti-murine Fcγ (JacksonImmunoResearch) was added to cells and they were incubated on ice for 30min. Cells were washed with FACS buffer and resuspended in 100 μl FACSbuffer for analysis.

14 clones were positive for binding to both human and cyno TfR (FIGS. 2Aand 2B). These clones were further subcloned and evaluated for bindingto both human and cyno TfR by ELISA, and epitope mapped on huTfR usingthe apical binding phage clone identified above. Briefly, the apicaldomain phage competition ELISA was performed in maxisorp plates coatedwith 2 μg/ml of purified human or cyno TfR in PBS at 4° C. overnight.Plates were washed with PBS/0.05% Tween 20 and blocked using Superblockwith casein (Thermo Scientific, Hudson, N.H.). A 30 μl aliquot ofhybridoma supernatant (normalized to 10 μg/ml) was added to each wellfor 45 min. This was followed by the addition of 30 μl apicaldomain-binding phage at OD 0.05 for 15 min. Plates were washed withPBS/0.05% Tween 20 and 1:1000 diluted HRP-Mouse-anti M13 (GE healthcare)was added the plate and incubated for 1 h at room temperature. Plateswere washed with PBS/0.05% Tween 20 and bound phage were detected usingTMB substrate (BioFX Laboratories, Owings Mills). Nine of the fourteenclones were found to block binding of the apical binding antibodydisplayed on phage (see FIG. 2C).

Antibody affinities were measured using surface plasmon resonance(“SPR”) (Biacore™, GE Healthcare). Anti-His antibody (Qiagen) wascoupled onto four different flow cells of a BIACORE™ CM5 sensor chip(Biacore, Inc., Piscataway, N.J.) at between 6000 and 8000 RUImmobilization was achieved by random coupling through amino groupsusing a protocol provided by the manufacturer. 10×HBS-P (Biacore, Inc.,Piscataway, N.J.) was diluted in water and served as the dilution andrunning buffer. Purified human or cyno TfR was captured, followed by a3-fold dilution series of IgG or Fab that was injected at a flow rate of30 ml/min using the single cycle kinetics method. Affinity constantswere determined using a simple 1:1 Langmuir binding model or using asteady state model when k_(on) or k_(off) was beyond the detectionlimit. The equilibrium dissociation constant (K_(D)) was calculated asthe ratio of association rate constant (k_(on)) and dissociation rateconstant (k_(off)). The results are shown in FIG. 2C.

Each hybridoma was cloned. Total RNA was isolated from hybridoma usingan RNeasy mini kit (Qiagen). cDNA was generated using a SMART 5′ RACEcDNA Amplification kit (Clontech) based on the manufacturer'sinstructions. The variable region of each antibody was amplified usingUPM (5′ oligo) provided in the kit and a 3′ oligo that anneals to theconstant region. The entire PCR product was then cloned intopCR4Blunt-TOPO vector (Invitrogen) for sequencing. After sequenceanalysis, the hybridomas could be further subdivided into 4 groups(FIGS. 3A-3D). Clones that competed with the apical binding antibodyfell into 3 related sequence classes (FIG. 3 A-C). The 4 non-apicalclones (FIG. 3D) consisted of 2 related clones and 2 other uniquesequences. The light and heavy chain CDRs of each clone are provided inTable 3.

TABLE 3Light and Heavy Chain CDRs of Cross-Reactive Anti-Cyno/Human TfR AntibodiesSEQ SEQ SEQ Clone Heavy/ ID ID ID name Light HVR1 # HVR2 # HVR3 # 7A4Light RASESVDSYGNSFMH  29 RASNLES  30 QQSNEAPPT  31 Heavy DYAMH  32GISTYFGRTNYNQKFKG  33 GLSGNYVMDY  34 8A2 Light RASESVDSYGNSFMH  35RASNLES  30 QQSNEGPPT  36 Heavy DYGMH  37 VISPYSGRTNYNQNFKG  38GLSGNYVVDY  39 15D2 Light RASESVDSYGNSFMH  35 RASNLES  30 QQSNEGPPT  36Heavy DYAMH  32 VISFYSGKTNYNQKFMG  40 GLSGNYVMDY  34 10D11 LightRASESVDSYGNSFMH  41 RASNLES  30 QHSNEDPPT  42 Heavy DYGMH  37VISPYSGKTNYSQKFKG  43 GLSGNFVMDF  44 7B10 Light RASESVDSYGNSFMH  29RASNLES  30 QQSNEAPPT  31 Heavy DYAMH  32 GISTYFGRTNYNQKFKG  33GLSGNYVMDY  34 Consensus RASESVD(S/D)YG(N/P)  45 RASNLES  30Q(Q/H)SNE(A/G/D)PPT  46 Class I Light SFMH CDRs Consensus DY(A/G)MH  47(G/V)IS(T/F/P)Y(F/S)G(R/K)  48 GLSGN(Y/F)V(M/V)D(Y/F)  49 Class I heavyTNY(N/S)Q(K/N)F(K/M)G CDRs 15G11 Light RASDNLYSNLA  50 DATNLAD  51QHFWGTPLT  52 Heavy SYWMH  53 EINPTNGRTNYIEKFKS  54 GTRAYHY  55 16G5Light RASENIYSNLA  56 AATDLAD  57 QHFWGTPLT  52 Heavy SYWMH  53EINPTNGRTNYNENFKS  58 GTRAYHF  59 13C3 Light RASDNIYSNLA  60 AATNLAD  61QHFWGTPLM  62 Heavy SYWMH  53 EINPINGRTNYSEKFKK  63 GTRAYHY  55 16G4Light RASDNIYSNLA  60 AVTNLAD  64 QHFWGTPLT  52 Heavy SYWMH  53EINPSNGRTNYNETFKS  65 GTRAYHY  55 Consensus RAS(E/D)N(L/I)YSNLA  66(D/A)(A/V)T(N/D)LAD  67 QHFWGTPL(T/M)  68 Class II Light CDRs ConsensusSYWMH  53 EINP(T/I/S)NGRTNY(I/N/S)E  69 GTRAYH(Y/F)  70 Class II Heavy(K/N/T)FK(S/K) CDRs 16F6 Light RASKSISKYLA  71 SGSTLQS  72 QQHNEYPWT  73Heavy SEYAWN  74 YISYSGTTSYNPSLKS  75 YGYGNPATRYFDV  76 7G7 LightRARQSVSTSSYSFMH  77 YASIQES  78 QHTWEIPFT  79 Heavy SYWMH  80NIYPGSGSTKYDERFKS  81 GGYDSRAWFAY  82 4C2 Light RARQSVSTSSYSFMH  77YASIQES  78 QHTWEIPFT  79 Heavy SYWMH  80 NIYPGSGSTKYDEKFKS  83GGYDSRAWFAH  84 1B12 Light TTSSSVPSSYFH  85 STSNLAS  86 HQYHRSPFT  87Heavy DYYMY  88 SISNGGDNTYYPDTVKG  89 QGALYDGYYRGAMDY  90 13D4 LightRAGQDITNYLN  91 YTSRLHS  92 QQANTLPYT  93 Heavy NYWIE  94EILPGSGSTKYNEKFKG  95 RGGYGYDGEFAY  96 Consensus (R/T)(A/T)(R/S/G)  97(Y/S)(A/T)S(I/N/R)(Q/L)  98 (Q/H)(H/Q)(T/Y/A)(W/  99 Class IV Light(Q/S)(S/-)(V/-)(S/-) (E/A/H)S H/N)(E/R/T)(I/S/L)P CDRs(T/-)(S/V/D)(S/P/I) (F/Y)T (Y/S/T)(S/N)(F/Y) (M/F/L)(H/N) Consensus(S/D/N)Y(W/Y)(M/I) 100 (N/S/E)I(Y/S/L)(P/N)G(S/G) 101(G/Q/R)G(Y/A/G)(D/L/Y) 102 Class IV) (H/Y/E) (G/D)(S/N)T(K/Y)Y(D/P/N)(S/Y/G)(R/D/Y)(A/G/D) Heavy CDRs (E/D)(R/K/T)(F/V)K(S/G)(W/Y/G)(F/Y/E)(R/F/-) (G/-)(A/-)(M/-)(A/D) (Y/H)

Representative clones from each class, (15G11, 7A4, 16F6 and 7G7), areexemplified herein for humanization and further characterization.Humanization was achieved using HVR grafts along with the inclusion ofselect vernier positions as outlined below and FIGS. 4A-4E. 15G11 washumanized by grafting the HVRs into the IGKV1-NL1*01 and IGHV1-3*01human variable domains. Combinations of different mouse vernierpositions were included in the humanized variants as outlined in FIG.4E. Humanized 15G11 variant 15G11.v5 contains selected vernier positionsin VL (positions 43 and 48) and VH (positions 48, 67, 69, 71 and 73) asoutlined in FIG. 4A. In addition, The N-terminus of VH was changed fromQ to E. For humanization of 7A4, an HVR graft was made using the 7A4heavy chain and 8A2 light chain HVRs (7A4 and 8A2 are related clones,FIG. 3A). HVRs were grafted into the IGKV4-1*01 and IGHV1-2*02 humanvariable domains. Combinations of different mouse vernier positions wereincluded in the humanized variants as outlined in FIG. 4E. Humanized 7A4variant, 7A4.v15 contains selected vernier positions in VL (position 68)and VH (positions 24 and 71) and the CDR-L3 change G94A, as outlined inFIG. 4B. 7G7 was humanized by grafting the HVRs into the kappa 4 andsubgroup I human consensus variable domains along with selected vernierpositions in VH (position 93) as outlined in FIG. 4C. This humanizedvariant is called 7G7.v1. 16F6 was humanized by grafting the HVRs intothe IGKV1-9*01 and IGHV4-59*01 human variable domains. Combinations ofdifferent mouse vernier positions were included in the humanizedvariants as outlined in FIG. 4E. Humanized 16F6 variant 16F6.v4 contains2 changes in VL (I48L and F71Y) as well as selected vernier positions inVL (positions 43 and 44) and VH (positions 71 and 78) as outlined inFIG. 4D.

TABLE 4 Light and Heavy Chain CDRs of Humanized Antibodies/Fabs SEQ SEQSEQ Clone Heavy/ ID ID ID name Light CDR1 # CDR2 # CDR3 # 15G11.v5 LightRASDNLYSNLA 50 DATNLAD 51 QHFWGTPLT  52 Heavy SYWMH 53 EINPTNGRTNYIEKFKS54 GTRAYHY  55 7A4.v15 Light RASESVDSYGNSFMH 29 RASNLES 30 QQSNEAPPT 127Heavy DYAMH 32 GISTYFGRTNYNQKFKG 33 GLSGNYVMDY  34 7G7.v1 LightRARQSVSTSSYSFMH 77 YASIQES 78 QHTWEIPFT  79 Heavy SYWMH 80NIYPGSGSTKYDERFKS 81 GGYDSRAWFAY  82 16F 6.v4 Light RASKSISKYLA 71SGSTLQS 72 QQHNEYPWT  73 Heavy SEYAWN 74 YISYSGTTSYNPSLKS 75YGYGNPATRYFDV  76

The affinity of humanized variants for human and cyno TfR was determinedby SPR as IgG (FIG. 4E). Selected clones were also analyzed by SPR asFab to assess monovalent affinity (Table 7). In both cases, the SPRexperiments were performed as described above.

TABLE 5 Biacore Binding Data for Selected Fab-Formatted Variants HuTfRCynoTfR Cy/hu Sample Ka Kd KD Ka Kd KD ratio Mu15G11.Fab 1.38E+064.65E−03 3.37E−09 1.07E+06 6.23E−03 5.81E−09 1.72 Mu15G11.Fab 6.34E+051.52E−03 2.41E−09 4.85E+05 3.68E−03 7.57E−09 3.15 Hu15G11.v1.Fab6.38E+05 0.006986 1.09E−08 5.05E+05 0.0373  7.39E−08 Hu15G11.v3.Fab6.42E+05 0.004657 7.26E−09 4.83E+05 0.0201  1.09E−08 Hu15G11.v5.Fab4.56E+05 0.004063 8.91E−09 hu15G11.v5.Fab 7.76E+05 0.003643 4.70E−091.41E+06 0.02184  1.56E−08 3.4 Mu7A4.Fab 1.65E+06 3.13E−04 1.90E−101.14E+06 8.45E−04 7.41E−10 3.9 Hu7A4.v5.Fab 2.24E+06 1.53E−03 6.86E−101.18E+06 6.41E−03 5.44E−09 Hu7A4.v8.Fab 9.28E+05 1.07E−03 1.15E−097.97E+05 6.81E−03 8.55E−09 Hu7A4.v9.Fab 1.71E+06 6.86E−04 4.01E−108.08E+05 3.42E−03 4.23E−09 Hu7A4.v12.Fab 3.32E+06 8.44E−04 2.55E−101.74E+06 3.31E−03 1.90E−09 hu7A4.v15.Fab 9.10E+05 3.17E−04 3.48E−103.78E+05 0.001618 4.28E−09 11 Hu7G7.v1 Fab 1.44E+05 0.006594 4.58E−083.84E+04 0.007231 1.88E−07 4.4 Mu16F6.Fab 6.07E+04 1.90E−04 3.13E−095.11E+04 1.37E−03 2.68E−08 8.56 Hu16F6.v4.Fab 1.31E+05 1.69E−04 1.29E−099.89E+04 2.44E−03 2.47E−08 19.1

The binding epitope of the antibodies were re-confirmed as follows. ATf-TfR blocking ELISA was performed in maxisorp plates coated with 2μg/ml of purified human TfR in PBS at 4° C. overnight. Plates werewashed with PBS/0.05% Tween 20 and blocked using Superblock blockingbuffer in PBS (Thermo Scientific, Hudson, N.H.). 50 μl of 12.5 μM humanholo-Tf (R&D Systems, Minneapolis, Minn.) was added to the plates for 40min. A 50 μl titration of hu7A4.v15, hu15G11.v5, Tf competitingantibody, and hu7G7.v1 (beginning at 10 μg/ml, 1:3 serial dilution) wasadded to the plate and incubated for 20 min. Plates were washed withPBS/0.05% Tween 20 and 1:1000 diluted HRP-Goat-anti human Fcγ (JacksonImmunoResearch) was added to the plate and incubated for 1 h at roomtemperature. Plates were washed with PBS/0.05% Tween 20 and detectedusing TMB substrate (BioFX Laboratories, Owings Mills).

An HFE-TfR binding ELISA was performed in maxisorp plates coated with 1μg/ml of HFE in PBS at 4° C. overnight. Plates were washed withPBS/0.05% Tween 20 and blocked using Superblock blocking buffer in PBS(Thermo Scientific, Hudson, N.H.). A titration of human TfR (start at100 μg/ml, 1:3 serial dilution) was added to the plate and incubated for1 h. 1 μg/ml of hu15G11.v5, hu7A4.v15 or hu7G7.v1 was then added to theplate for 1 h. Plates were washed with PBS/0.05% Tween 20 and 1:1000diluted HRP-Goat-anti human Fcγ (Jackson ImmunoResearch) was added theplate and incubated for 1 h at room temperature. Plates were washed withPBS/0.05% Tween 20 and detected using TMB substrate (BioFX Laboratories,Owings Mills). An HFE-TfR blocking ELISA was performed in maxisorpplates coated with 1 μg/ml HFE in PBS at 4° C. overnight. Plates werewashed with PBS/0.05% Tween 20 and blocked using Superblock blockingbuffer in PBS (Thermo Scientific, Hudson, N.H.). In a NUNC™ plate, atitration of hu7A4.v15, hu15G11.v5, Tf competiting antibody, humanholo-Tf and control IgG (400 μg for all antibody, 8000 μg/ml for holotransferrin, 1:3 serial dilution) was combined with 2 μg/ml ofbiotinylated human TfR and incubated for 1 h. The mixture was then addedto the HFE coated plate for 1 h at room temperature. Plates were washedwith PBS/0.05% Tween 20 and 1:1000 diluted HRP-streptavidin(SouthernBiotech, Birmingham) was added the plate and incubated for 1hour at room temperature. Plates were washed with PBS/0.05% Tween 20 andbiotinylated human TfR bound to the plate was detected using TMBsubstrate (BioFX Laboratories, Owings Mills).

Binding of these humanized variants to TfR was unaffected by thepresence of 6.3 μM holo-Tf, whereas the binding of the Tf competingantibody that binds to the Tf binding site on TfR was inhibited (FIG.5). Further, humanized 7A4.v15, 15G11.v5 and 7G7.v1 could still bind HFEcaptured huTfR, indicating that they did not affect binding of huTfR toimmobilized HFE (FIG. 6A). In a related experiment, 7A4.v15 and 15G11.v5did not block biotinylated TfR from binding to immobilized HFE. Incontrast, this interaction was blocked by the Tf competing antibody andholo Tf (FIG. 6B). HFE and Tf are known to share a similar epitope onTfR (Bennet et al, Nature (2000) 403, 46-53).

Immobilized 15G11v.5 and anti-TfR^(C12) were evaluated for binding tobiotinylated human TfR ECD or monovalent M13 phage displaying the humanTfR apical domain. Anti-TfR^(C12) was derived from a synthetic antibodyphage library that was panned against human TfR ECD and binds to a siteon the human TfR which competes with transferrin binding. Antibodieswere coated at 1 μg/ml in PBS on Maxisorp plates. Bound biotinylatedhuman TfR ECD or TfR-apical domain phage were detected withHRP-streptavidin (GE health care, RPN 4401V) or HRP-anti-M13 (GE healthcare, 27-9421-01), respectively. FIG. 25 shows that 15G11v.5 binds tohuman TfR apical domain. The 15G11v.5 binding site was mapped to theapical domain, a site distant from the TfR ligand binding sites.

Example 2: Affinity Engineering Human/Cyno Cross-Reactive Anti-TfRAntibodies

In addition to the humanized variants described above, additionalaffinity engineered variants were made. Exemplified herein is affinityengineering of 15G11.v5 and 7A4.v15. Affinity variants were generated bymaking individual alanine substitutions in CDR-L3 or CDR-H3 usingstandard techniques. These variants were screened as IgG by ELISA andSPR to identify positions important for binding to human and cyno TfR;the monovalent affinity of selected variants as Fab was also determined.Ala scan variant IgGs or Fab were expressed in 293 cells and binding tohuman or cyno TfR quantified by ELISA in maxisorp plates coated with 1.8μg/ml of goat anti-human Fcγ (Jackson ImmunoResearch) in PBS at 4° C.overnight. Plates were washed with PBS/0.05% Tween 20 and blocked usingSuperblock blocking buffer in PBS (Thermo Scientific, Hudson, N.H.).Supernatants containing expressed IgG were diluted serially 1:5 andadded to the plate for 1 h. Purified hu15G11.v5 or hu7A4.v15 were usedas standards (diluted 1:5 beginning at 1 μg/ml). Plates were washed withPBS/0.05% Tween 20 and 1:1000 diluted HRP-Goat-anti kappa (SouthernBiotech) was added the plate and incubated for 1 h at room temperature.Plates were washed with PBS/0.05% Tween 20 and detected using TMBsubstrate (BioFX Laboratories, Owings Mills). Binding was also assessedby SPR, as described above. The results are shown in FIGS. 7A (15G11.v5variants) and 7B (7A4.v15 variants).

Further variants of 15G11.v5 with individual alanine substitutions atpositions in CDR-L1, CDR-L2, CDR-H1 and CDR-H2 were also generated,expressed and first screened for binding to human and cyno TfR by ELISA(Table 6). The Hu/Cy binding ELISA was performed in maxisorp platescoated with 2 μg/ml of purified human or cyno TfR in PBS at 4° C.overnight. Plates were washed with PBS/0.05% Tween 20 and blocked usingSuperblock blocking buffer in PBS (Thermo Scientific, Hudson, N.H.).Cell culture supernatants containing the expressed Ala scan variant IgGswere serially diluted 1:5 and added to the wells for 1 h. Plates werewashed with PBS/0.05% Tween 20 and 1:1000 diluted HRP-Goat-anti humanFcγ (Jackson ImmunoResearch) was added the plate and incubated for 1hour at room temperature. Plates were washed with PBS/0.05% Tween 20 anddetected using TMB substrate (BioFX Laboratories, Owings Mills).

TABLE 6 ELISA Analysis of hu15G11.v5 IgG Ala Variants CynoTfR HuTfR EC50(ng/ml) EC50 (ng/ml) 15G11.v5 1.8 0.8 R- G26A 6.1 1.1 Y27A 1467.1 21.1T28A 0.8 0.4 F29A 12.5 1.5 T30A 0.9 0.4 S31A 0.1 0.05 Y32A 1.4 0.4 W33A362.5 59.6 M34A 1.1 0.4 HVR-H2 G49A 1.1 0.3 E50A 409.7 20.6 I51A 0.6 0.2N52A 0.9 0.3 P52aA 8.1 3.9 T53A 0.7 0.3 R56A 5405.4 55.1 N58A 80.4 6.3Y59A 0.7 0.3 I60A 0.7 0.3 E61A 0.6 0.2 K62A 0.7 0.3 F63A 0.6 0.4 K64A0.6 0.3 S65A 0.7 0.2 HVRL1 R24A 0.4 0.1 S26A 0.6 0.2 D27A 0.8 0.2 N28A0.8 0.2 L29A 0.9 0.3 Y30A 1.0 0.3 S31A 0.7 0.3 N32A 4.0 1.6 L33A 0.10.05 HVR-L2 D50A 0.5 0.2 T52A 0.3 0.2 N53A 0.6 0.3 L54A 0.5 0.4 D56A 0.50.3

Selected variants were then purified and their monovalent affinity forhuman or cyno TfR assessed by SPR (Table 7).

TABLE 7 Monovalent SPR Analysis of Select 15G11.v5 Fab Alanine VariantsHuTfR CynoTfR Cy/hu Ka Kd KD Ka Kd KD ratio Hu15G11.v5 Fab 6.74E+054.74E−03 7.03E−09 4.51E+05 1.27E−02 2.82E−08 4.0 Hu15G11.HC32A Fab2.38E+05 1.78E−03 7.47E−09 1.77E+05 8.51E−03 4.80E−08 6.4 Hu15G11.HC34AFab 5.64E+05 4.03E−03 7.14E−09 3.04E+05 9.90E−03 3.26E−08 4.6Hu15G11.HC52A Fab 5.30E+05 2.36E−02 4.44E−08 4.87E+05 5.67E−02 1.17E−072.6 Hu15G11.HC52A Fab 5.64E+05 1.96E−02 3.46E−08 ND ND ND NDHu15G11.HC51A Fab 4.33E+05 1.04E−02 2.39E−08 3.13E+05 3.29E−02 1.05E−074.4 Hu15G11.HC53A Fab 8.90E+05 1.15E−02 1.29E−08 4.84E+05 2.50E−025.18E−08 4.0 Hu15G11.HC54A Fab 2.06E+05 8.71E−03 4.24E−08 2.80E+051.69E−02 6.02E−08 1.4

Example 3: Bispecific Anti-Human TfR Antibody Construction and IN VIVOAnalysis

Certain of the foregoing antibody variants were reformatted asbispecific antibodies with a second arm specifically binding to BACE1.The anti-human TfR antibodies Hu15G11.v5, Hu15G11.LC92A, Hu15G11.HC52Aand Hu15G11.HC53A were used to engineer the TfR binding arm of thebispecific using ‘knob in hole’ bispecific antibody constructiontechnology (Carter, P. (2001) J. Immunol Methods 248, 7-15; Ridgway, J.B., Presta, L. G., and Carter, P. (1996) Protein Eng. 9, 617-621;Merchant, A. M., Zhu, Z., Yuan, J. Q., Goddard, A., Adams, C. W.,Presta, L. G., and Carter, P. (1998) Nat. Biotechnol. 16, 677-681;Atwell, S., Ridgway, J. B., Wells, J. A., and Carter, P. (1997) J. Mol.Biol. 270, 26-35). In addition to the knob and hole mutations in the Fcfor anti-TfR (hole) and anti-BACE1 (knob), all half-antibodies containedmutations in the Fc region that abrogated effector function (N297G) andHu15G11.v5 and Hu15G11.LC92A contained an additional Fc mutation thatabrogated effector function (D265A). The knob and hole half-antibodieswere purified separately from E. coli and combined at a 1:1.1 ratio ofanti-TfR to prevent formation of anti-TfR homodimers. Assembly of thebispecific antibody was completed by reductive annealing for at leastthree days at room temperature in a buffer containing reducedglutathione at a 100x ratio to antibody and 200 mM arginine pH 8.0.Following assembly, bispecific antibodies were purified by hydrophobicinteraction chromatography. The assembly was confirmed by liquidchromatography mass spectroscopy and SDS-PAGE. The purified antibodieswere confirmed to be homogeneous and monodisperse by size exclusion andmulti angle laser light spectroscopy.

The resulting bispecific antibodies were called 15G11.v5 (anti-TfR³),15G11.W92A (15G11.LC92A or anti-TfR²), Hu15G11.N52A (anti-TfR^(52A)) andHu15G11.T53A (anti-TfR^(53A)). The monovalent affinity and kinetics forhuman and cyno TfR was determined for 115G11.v5 and 115G11.W92A by SPR,as above (see Table 9). Anti-TfR¹ and anti-TfR² possess similarmonovalent binding affinities as anti-TfR^(A) and anti-TfR^(D) do forbinding to mouse TfR (see Atwal et al., Sci. Transl. Med. 3, 84ra43(2011); Yu et al., Sci. Transl. Med. 25 May 2011: Vol. 3, Issue 84, p.84ra44).

TABLE 8 Monovalent SPR Analysis of 15G11.v5 (TfR¹) and 15G11.W92A(LC92A, TfR²) Cyno/ HuTfR CynoTfR human Ka Kd KD Ka Kd KD ratioHu15G11.v5 Fab 6.74E+05 4.74E−03 7.03E−09 4.51E+05 1.27E−02 2.82E−08 4.0Hu15G11.W92A 1.28E+05 3.77E−02 2.95E−07 8.36E+04 5.20E−02 6.22E−07 2.1Bispecific

Additionally, the binding affinity of the anti-TfR¹, anti-TfR²,Hu15G11.N52A and Hu15G11.T53A bispecific antibodies were measuredagainst human and cyno TfR by SPR as previously described. As shown inTable 9 below, Anti-TfR^(52A) and anti-TfR^(53A) have binding affinitiesto human and cyno TfR between TfR1^(h15G11.v5) and TfR2^(LC92A).

TABLE 9 Anti-cyno/human TfR antibodies (nM) Human TfR Cyno TfRTfR1^(h15G11.v5) 10 37 TfR2^(LC92A) 270 810 TfR^(52A) 52 343 TfR^(53A)24 143

Example 4: Impact of Effector-Containing and Effectorless Monospecificand Bispecific Antibodies on a Human Erythroleukemia Cell Line andPrimary Bone Marrow Mononuclear Cells

Prior studies in mice had determined that antibodies binding murine TfRwith effector function and/or complement binding capabilitiesselectively depleted TfR-expressing reticulocytes. To ascertain whetherthe depletion observed in the mouse studies was unique to a murinesystem, further experiments were performed utilizing anti-TfR that bindto human TfR.

ADCC assays were carried out using peripheral blood mononuclear cells(PBMCs) from healthy human donors as effector cells. A humanerythroleukemia cell line (HEL, ATCC) and primary human bone marrowmononuclear cells (AllCells, Inc.) were used as target cells. Tominimize inter-donor variability which could potentially arise fromallotypic differences at the residue 158 position in FcγRIIIA, blooddonors were limited to those carrying the heterozygous RcγRIIIA genotype(F/V158) in the first set of experiments (FIG. 8A-B). For the second setof experiments (FIG. 9A-B), only HEL cells were used as the targetcells, with PBMCs from healthy human donors carrying either the F/V158genotype or the FcγRIIIA V/V158 genotype. The V/V158 genotype was alsoincluded in this assay due to the known association with increased NKcell-mediated ADCC activity as well as ability to bind IgG4 antibodies(Bowles and Weiner, 2005; Bruhns et al. 2008). Cells were counted andviability was determined by Vi-CELL® (Beckman Coulter; Fullerton,Calif.) following the manufacturer's instructions.

PBMCs were isolated by density gradient centrifugation using Uni-Sep™blood separation tubes (Accurate Chemical & Scientific Corp.; Westbury,N.Y.). Target cells in 50 μL of assay medium (RPMI-1640 with 1% BSA and100 units/mL penicillin and streptomycin) were seeded in a 96-well,round-bottom plate at 4×10⁴/well. Serial dilutions of test and controlantibodies (50 μL/well) were added to the plates containing the targetcells, followed by incubation at 37° C. with 5% CO₂ for 30 minutes toallow opsonization. The final concentrations of antibodies ranged from0.0051 to 10,000 ng/mL following 5-fold serial dilutions for a total of10 data points. After the incubation, 1.0×10⁶ PBMC effector cells in 100μL of assay medium were added to each well to give a ratio of 25:1effector: target cells, and the plates were incubated for an additional4 hours. The plates were centrifuged at the end of incubation and thesupernatants were tested for lactate dehydrogenase (LDH) activity usinga Cytotoxicity Detection Kit™ (Roche Applied Scinece; Indianapolis,Ind.). The LDH reaction mixture was added to the supernatants and theplates were incubated at room temperature for 15 minutes with constantshaking. The reaction was terminated with 1 M H₃PO₄, and absorbance wasmeasured at 490 nm (the background, measured at 650 nm was subtractedfor each well) using a SpectraMax Plus microplate reader. Absorbance ofwells containing only the target cells served as the control for thebackground (low control), whereas wells containing target cells lysedwith Triton-X100 provided the maximum signal available (high control).Antibody-independent cellular cytotoxicity (AICC) was measured in wellscontaining target and effector cells without the addition of antibody.The extent of specific ADCC was calculated as follows:

${\%\mspace{14mu} A\; D\; C\; C} = {100 \times \frac{{A_{490}\mspace{14mu}({Sample})} - {A_{490}\mspace{11mu}\left( {A\; I\; C\; C} \right)}}{{A_{490}\mspace{14mu}\left( {{High}\mspace{14mu}{Control}} \right)} - {A_{490}\mspace{14mu}\left( {{Low}\mspace{14mu}{Control}} \right)}}}$ADCC values of sample dilutions were plotted against the antibodyconcentration, and the dose-response curves were fitted to afour-parameter model using SoftMax Pro.

In a first set of experiments, the ADCC activity of various anti-humanTfR constructs were assessed using either a human erythroleukemia cellline (HEL cells) or primary human bone marrow mononuclear cells as thetarget cells. Bivalent IgG1 effector function-competent anti-human TfR1antibody 15G11, and a bispecific form of this antibody with ananti-BACE1 arm in a human IgG1 format containing D265A and N297Gmutations to abrogate effector function (see Example 3), were tested atvarious concentrations in the ADCC assay, using anti-gD WT IgG1 as anegative control and murine anti-human HLA (class I) as a positivecontrol. The results are shown in FIGS. 8A and 8B. With either the HELcells as targets (FIG. 8A) or the bone marrow mononuclear cells astargets (FIG. 8B), the monospecific, effector positive anti-human TfRantibody 15G11 elicited significant ADCC activity. This activity wassimilar to that of the positive control anti-human HLA antibodies on theHEL cells, and at a robust yet lower level relative to the positivecontrol on the bone marrow mononuclear cells. The somewhat lower levelobserved in the bone marrow mononuclear cells experiment is likely dueto the fact that only a portion of the heterogeneous mixture of myeloidand erythroid lineage PBMC cells used in the experiment express highlevels of TfR, whereas the HEL cells have consistently high TfRexpression throughout the clonal cell population. In sharp contrast, thebispecific effectorless anti-humanTfR/BACE1 antibody did not display anyADCC activity in either HEL or bone marrow mononuclear cells, similar tothe negative control.

In a second set of experiments, the impact of switching the antibodyisotype in this assay system was assessed. The ADCC assay procedure wasidentical to that described above, with the exception that all targetcells were HEL cells, and the effector cells were PBMCs from healthyhuman donors either carrying the heterozygous FcγRIIIa-V/F158 genotypeor the homozygous FcγRIIIa-V/V158 genotype. All anti-human TfR testedwere bispecific with anti-gD, on three different Ig backbones: wild-typehuman IgG1, human IgG1 with the N297G mutation, and human IgG4. Ananti-Abeta antibody with a human IgG4 backbone was also tested, andmouse anti-human HLA (class I) served as a positive control. The resultsare shown in FIGS. 9A and 9B. As anticipated based on the knownassociation between effector cell activation and the V/V158 genotype(Bowles and Weiner 2005), ADCC activity was more robustly elicited byV/V158 donor PBMCs (˜45% of target cells impacted) relative to F/V158donors (˜25% of target cells impacted) (compare FIG. 9A to FIG. 9B).Anti-TfR/gD with the wild-type IgG1 induced robust ADCC in HEL cells,while the anti-TfR/gD with the effectorless IgG1 did not show any ADCCactivity in HEL cells, replicating the results from the first set ofexperiments. Notably, at concentrations of 100 ng/mL or higher,anti-TfR/gD of the IgG4 isotype showed a mild ADCC activity. Thisactivity was not observed in the anti-Abeta IgG4 results, indicatingthat TfR binding was required for the ADCC activity. This findingcorrelates with previous reports that IgG4 has minimal, but measurable,effector function (Adolffson et al., J. Neurosci. 32(28):9677-9689(2012); van der Zee et al. Clin Exp. Immunol 64: 415-422 (1986)); Tao etal., J. Exp. Med. 173:1025-1028 (1991)).

Example 5: Assessment of Bispecifie Anti-Human TrR/BACE1 BispecificAntibodies In Vivo

A. Pharmacokinetic, Pharmacodynamic and Safety Study

To evaluate the drug concentrations, pharmacodynamics effects, andsafety of the bispecific anti-human TfR antibodies in vivo, cynomolgusmonkeys (Macaca fascicularis) were dosed with bispecific antibodiesusing anti-TfR antibody clone 15G11 paired with the same anti-BACE1 armused in prior examples (anti-TfR¹/BACE1), or clone 15G11.LC92A pairedwith the same anti-BACE1 arm used in prior examples (anti-TfR²/BACE1) orHu15G11.N52A (anti-TfR^(52A)/BACE1) and Hu15G11.T^(53A)(anti-TfR^(53A)/BACE1). These bispecific antibodies were in a human IgG1format with N297G or D265A and N297G mutations abrogating effectorfunction, as described previously. As a control, an anti-gD molecule onhuman IgG1 was used. This study was performed in non-human primatesbecause crossreactivity of these anti-TfR antibodies is limited tonon-human primates and humans. In addition, studies have shown that themechanisms of drug transport between the cerebrospinal fluid (CSF) andplasma compartments may be similar between humans and primates (Poplacket al, 1977). The antibodies were administered by a single intravenous(IV) bolus injection into the saphenous vein at a dose of 30 mg/kg toconscious cynomolgus monkeys with indwelling cisterna magna catheters.At various timepoints up to 60 days post-dose, plasma, serum, and (CSF)were sampled. Sample analysis included hematology (whole blood),clinical chemistry (serum), antibody concentrations (serum and CSF), andpharmacodynamic response to the antibody (plasma and CSF). See FIG. 10for a detailed sampling scheme.

The concentrations of the dosed antibodies in cynomolgus monkey serumand CSF were measured with an ELISA using a sheep anti-human IgG monkeyabsorbed antibody coat, followed by adding serum samples starting at adilution of 1:100, and finished by adding a goat anti-human IgG antibodyconjugated to horseradish peroxidase monkey adsorbed for detection. Theassay had a standard curve range of 0.78-50 ng/mL and a limit ofdetection of 0.08 μg/mL. Results below this limit of detection werereported as less than reportable (LTR).

FIGS. 11A-B shows the results of the pharmacokinetic analysis foranti-TfR1/BACE1 and anti-TfR2/BACE1. The pharmacokinetic profile foranti-gD was as expected for a typical human IgG1 antibody in cynomolgusmonkey with a mean clearance of 3.98 mL/day/kg. Both anti-TfR/BACE1antibodies cleared faster than anti-gD, likely due to peripheraltarget-mediated clearance. Anti-TfR1/BACE1 had the fastest clearance,consistent with it having the highest binding affinity to TfR, whereasanti-TfR2/BACE1 showed an improved pharmacokinetic profile (ie,prolonged exposure in serum) as compared to anti-TfR1/BACE1, likely dueto its reduced affinity for TfR. The clearance for anti-TfR1/BACE1 andanti-TfR2/BACE1 were 18.9 mL/day/kg and 8.14 mL/day/kg, respectively.All antibodies were detected in the CSF at approximately oneone-thousandth of the serum concentration. However, there was highvariability, and overall no detectable difference in the CSF antibodyconcentrations across the molecules.

TABLE 10 Mean (±SD) PK parameter estimates for all test antibodiesfollowing a single IV bolus dose administration at 30 mg/kg incynomolgus monkeys (n = 5) AUC_(all) AUC_(inf) C_(max) CL V_(ss)Antibody (day*μg/mL) (day*μg/mL) (μg/mL) (mL/day/kg) (mL/kg) anti-gD 7640 ± 1790  7930 ± 1910 912 ± 141 3.98 ± 1.05 51.3 ± 10.2anti-TfR¹/BACE1 1610 ± 240 1610 ± 237 809 ± 132 18.9 ± 2.54 41.0 ± 8.18anti-TfR²/BACE1 3750 ± 528 3750 ± 530  850 ± 69.2 8.14 ± 1.21 41.2 ±6.06 SD = standard deviation; IV = intravenous; AUC_(all) = area underthe concentration-time curve from time 0 to the time of last measurableconcentration; AUC_(inf) = area under the concentration-time curveextrapolated to infinity; C_(max) = observed maximum serumconcentration; CL = clearance; V_(ss) = volume of distribution at steadystate; Min = minimum; Max = maximum.

FIG. 19 shows the results of the pharmacokinetic analysis foranti-TfR¹/BACE1, anti-TfR^(52A)/BACE1 and anti-TfR^(53A)/BACE1. Allanti-TfR/BACE1 antibodies cleared faster than anti-gD, likely due toperipheral target-mediated clearance. Anti-TfR¹/BACE1 had the fastestclearance, consistent with it having the highest binding affinity toTfR, whereas anti-TfR^(52A)/BACE1 and anti-TfR^(53A)/BACE1 showed animproved pharmacokinetic profile (ie, prolonged exposure in serum) ascompared to anti-TfR¹/BACE1, likely due to the reduced affinity for TfRof anti-TfR^(52A)/BACE1 and anti-TfR^(53A)/BACE1.

To look at the pharmacodynamic effect in response to anti-TfR/BACE1dosing, we measured Abeta₁₋₄₀ and sAPPα and sAPPβ levels in cynomolgusmonkey plasma and CSF. Abeta₁₋₄₀ was measured with an ELISA using ananti-Abeta₁₋₄₀ specific polyclonal antibody coat, followed by addingsamples, and finishing by adding a mouse anti-human Abeta¹⁻⁴⁰ monoclonalantibody conjugated to horseradish peroxidase for detection. The assayhas a limit of detection of 60 pg/mL for plasma and 140 pg/mL for CSF.Results below this concentration were reported as less than reportable(LTR). CSF concentrations of sAPPα and sAPPβ were determined using thesAPPα/sAPPβ Multi-spot assay (Mesoscale Discovery (Gaithersburg, Md.)).CSF was thawed on ice, then diluted 1:10 into 1% BSA in TBS-T (10 mMTris buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20). The assay wasperformed as per the manufacturer's protocol. The assay had lower limitof quantification values of 0.05 ng/ml for sAPPα and 0.03 ng/mL forsAPPβ.

FIGS. 12A-E summarize the pharmacodynamics behavior of the antibodies.In the periphery, plasma Abeta₁₋₄₀ levels remained unchanged followinganti-gD administration, but transiently decreased followinganti-TfR/BACE1 administration. Both variants reduced plasma Abeta₁₋₄₀levels, with a maximal inhibition of 50% achieved 1 day post-dosing.Plasma Abeta₁₋₄₀ levels gradually recovered, with animals givenanti-TfR¹/BACE1 returning to baseline Abeta₁₋₄₀ levels around 14 dayspost-dose. Abeta₁₋₄₀ levels returned to baseline levels between 21 and30 days post-dose in animals treated with anti-TfR²/BACE1. Bothanti-TfR/BACE1 antibodies reduced CSF Abeta₁₋₄₀ levels, with no changeobserved in anti-gD dosed animals. Anti-TrR¹/BACE1 administrationresulted in a more significant decrease in CSF Abeta₁₋₄₀ levels (averagemaximal inhibition 50% of baseline) than that of anti-TfR²/BACE1(average maximal inhibition 20% of baseline). sAPPβ production wasinhibited in anti-TfR/BACE1 treated animals, but not in animals whoreceived anti-gD. Similar to results for Aβ40, anti-TfR¹/BACE1 had astronger inhibitory effect on sAPPβ production than anti-TfR²/BACE1.sAPPα production was stimulated during BACE1 inhibition by bothanti-TfR¹/BACE1 and anti-TfR²/BACE1, and the response correlatedinversely with the level of inhibition observed for sAPPβ and Abeta₁₋₄₀.SAPPα and sAPPβ are the primary processing products of amyloid precursorprotein (APP), and their levels are highly correlated. The ratio ofsAPPβ/sAPPα normalizes the results to potential changes in basal APPexpression or potential preanalytical differences in CSF collection andhandling over the course of the study. The ratio of CSF sAPPβ/sAPPα withanti-TfR¹/BACE1 demonstrated a more robust PD effect thananti-TfR²/BACE1. Thus, these results support target (i.e. BACE1)engagement by the anti-TfR/BACE1 antibodies.

The PD response for anti-TfR^(52A)/BACE1 and anti-TfR^(53A)/BACE1 alsocorrelates with the duration of antibody exposure and a reduced affinityTfR arm shows increased reduction in Aβ₄₀ (data not shown). These dataalso support target engagement by these bispecific antibodies.

Overall, these results suggest that a bispecific anti-TfR/BACE1 antibodywith an affinity for human TfR between that of anti-TfR¹/BACE1 andanti-TfR²/BACE1 would likely have a desirablepharmacokinetic/pharmacodynamic balance.

No safety signals were observed in this study. There were no evidenteffects on any hematology or clinical chemistry parameters of monkeysgiven 30 mg/kg of any bispecific antibody administered up to 60 dayspost-dose. Importantly, reticulocyte levels were unaffected by treatmentwith either anti-TfR¹/BACE1 or anti-TfR²/BACE1 (FIG. 13), as expectedsince these antibodies were effector-function-impaired and the overalllevel of circulating early reticulocytes with high TfR levels is verylow in normal primates (see Example 4).

Example 6: Assessment of Bispecific Anti-Human TfR/BACE1 BispecificAntibodies In Vivo

To examine the relationship between antibody pharmacodynamics in CSF andpharmacokinetics in brain, cynomolgus monkeys (Macaca fascicularis) weredosed with bispecific antibodies anti-TfR¹/BACE1 or anti-TfR²/BACE1, asin the previous example. These bispecific antibodies were in a humanIgG1 format with D265A and N297G mutations abrogating effector function.As a control, an anti-gD molecule on human IgG1 was used. Forcomparison, we also dosed with a bivalent anti-BACE1 antibody, which isthe same clone used for the bispecific antbibodies. The antibodies wereadministered by a single intravenous (IV) bolus injection into thesaphenous vein at a dose of 30 mg/kg to conscious cynomolgus monkeyswith indwelling cisterna magna catheters. Baseline CSF samples werecollected 24 and 48 hours prior to dosing, and another CSF sample wascollected 24 hours post-dose (as shown schematically in FIG. 14).Following CSF collection 24 hours post-dose, animals were perfused withsaline and brains were harvested for analysis of antibodyconcentrations. Different brain regions were homogenized in 1% NP-40(Cal-Biochem) in PBS containing Complete Mini EDTA-free proteaseinhibitor cocktail tablets (Roche Diagnostics). Homogenized brainsamples were rotated at 4° C. for 1 hour before spinning at 14,000 rpmfor 20 minutes. The supernatant was isolated for brain antibodymeasurement, using the ELISA method described in the previous example.Blood was also collected to confirm peripheral exposure andpharmacodynamics responses, which were similar to our observations inExample 5.

The pharmacodynamic effects of anti-TfR¹/BACE1 and anti-TfR²/BACE1 asassessed in CSF were also similar to that observed in the previousexample. FIG. 15 demonstrates that the ratio of CSF sAPPβ/sAPPαdecreased robustly following dosing with anti-TfR¹/BACE1 Anti-TfR²/BACE1did not show an evident decrease at 24 hours post-dose in this study.Anti-BACE1 also showed no effect. Analysis of brain concentrations ofantibody revealed that both the control IgG and the anti-BACE1 antibodyhad limited uptake into the brain, at levels that were just abovedetection in our assay (average ˜670 pM). Anti-TfR²/BACE1 had ˜3-foldimproved brain uptake over control IgG (average ˜2 nM), andanti-TfR¹/BACE1 had the best brain uptake, ˜15-fold greater than controlIgG (average ˜10 nM). The brain antibody concentrations for thedifferent antibodies correlated with the pharmacodynamics response seenin CSF in our studies, with anti-TfR¹/BACE1 having the best brain uptakeand most robust pharmacodynamics effect, and anti-TfR²/BACE1 having lessbrain uptake and a more modest effect.

These results extend our previous findings to demonstrate thatTfR-binding bispecific antibodies improve uptake in brain of non-humanprimates. In primates, as in mice, there is likely an optimum affinityto TfR that best balances brain uptake and TfR-mediated clearance. Inour example, the higher affinity anti-TfR¹/BACE1 demonstrates good brainuptake, and is affected by peripheral target-mediated clearance. Thereduced affinity TfR²/BACE1 has improved clearance properties, butappears to have such low binding for TfR as to not be able to beefficiently transported by TfR (much in the same way that thelowest-affinity anti-TfR antibody TfR^(E) in US2012/0171120 passes someaffinity threshold beyond which the affinity is too low to permitsufficient interaction between the antibody and TfR such that theantibody would remain associated with TfR as TfR begins thetranslocation process). From the results of this experiment, ananti-human/cyno TfR/BACE1 bispecific antibody having affinity for TfRbetween that of TfR¹ and TfR² would be predicted to have improved uptakeand clearance properties over either anti-TfR¹/BACE1 or anti-TfR²/BACE1in this system.

Example 7: Creation of Additional Effectorless Mutations in the Contextof a Bispecific Transferrin Receptor Antibody

Other mutations in the Fc region, which abrogate effector function inaddition to N297G and D265A were tested for their ability to reduce orprevent depletion of TfR-expressing reticulocytes. Specifically, the Fcmutations L234A, L235A and P329G (“LALAPG”) which are described in USApplication Publication No 2012/0251531, which is incorporated herein byreference, were incorporated into the anti-TfR^(D)/BACE1 antibody (whichis described in International Application Publication No. WO2013/177062, and which is incorporated by reference herein in itsentirety).

Pharmacokinetic analysis and reticulocyte count following a singleantibody administration in mice were performed as follows. Wild typefemale C57B/6 mice ages 6-8 weeks were used for all studies. Theanimals' care was in accordance with institutional guidelines. Mice weredosed intravenously with a single 50 mg/kg dose of either an anti-gDantibody (murine IgG2a) with the LALAPG mutations, an anti-TfR^(D)/BACE1antibody (rat/murine chimera) with the LALAPG mutations. Total injectionvolume did not exceed 250 μL and antibodies were diluted in D-PBS whennecessary (Invitrogen). After 24 hours, whole blood was collected priorto perfusion in EDTA microtainer tubes (BD Diagnostics), allowed to sitfor 30 minutes at room temperature, and spun down at 5000× x g for 10minutes. The top layer of plasma was transferred to new tubes forantibody measurements.

Total antibody concentrations in mouse plasma was measured using ananti-mouse IgG2a (allotype a)/anti-mouse IgG2a (allotype a) ELISA. NUNC384-well Maxisorp immunoplates (Neptune, N.J.) were coated with mouseanti-mouse IgG2a allotype A, an allotype A specific antibody(BD/Phamigen San Jose, Calif.), overnight at 4° C. Plates were blockedwith PBS, 0.5% BSA for 1 hour at 25° C. Each antibody (anti-gD and theanti-TtR/BACE1 bispecific variants) was used as a standard to quantifyrespective antibody concentrations. Plates were washed with PBS, 0.05%Tween-20 using a microplate washer (Bio-Tek instruments, Inc., Winooski,Vt.), and standards and samples diluted in PBS containing 0.5% BSA, 0.35M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.2% BgG, 0.05% Twcen-20 and 15 ppmProclin® (Sigma-Aldrich) were added for two hours at 25° C. Boundantibody was detected with biotin-conjugated mouse anti-mouse IgG2aallotype A, an allotype A specific antibody (BD/Pharmigen San Jose,Calif.). Bound biotin-conjugated antibody was detected with horseradishperoxidase-conjugated streptavidin (GE Helathcare Life Sciences,Pittsburgh, Pa.). Samples were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, Md.) and absorbance measuredat 450 nm on a Multiskan Ascent reader (Thermo Scientific, Hudson,N.H.). Concentrations were determined from the standard curve using afour-parameter non-linear regression program. The assay had lower limitof quantification (LLOQ) values of 78.13 ng/ml in plasma. Statisticalanalysis of differences between experimental groups was performed usinga two-tailed unpaired t-test.

Upon administration of the anti-TfR^(D)/BACE1 antibodies containing theFc LALAPG mutations, the mice displayed no clinical symptoms as had beenpreviously observed using antibodies with full effector function. SeeCouch et al., Sci. Trans. Med. 5:183ra57 (2013). FIG. 20 shows theresults of the pharmacokinetic analysis.

Additionally, immature and total reticulocyte counts were determinedusing the Sysmex XT2000iV (Sysmex, Kobe, Japan) according tomanufacturer's instructions. At 24 hours post dose, there was noobserved difference in the immature reticulocyte fraction or the totalreticulocyte count with any antibody tested as seen in FIG. 21. Theseresults suggest that the LALAPG mutation not only abrogates antibodyeffector function but also reduces complement binding andcomplement-mediated reticulocyte clearance seen even with aneffectorless antibody framework (Couch et al. 2013). This is consistentwith another report that incorporation of the LALA mutation on a humanIgG1 can limit complement binding (Hessell et al. Nature 449:101-104(2007)).

Example 8: Creation of FcRNa^(HIGH) Bispecific Variants

In order to increase the half-life of the bispecific antibodies, andthereby potentially increase the concentration of the antibody in thebrain, bispecific variants were made containing mutations in the IgGconstant domain and specifically in the Fc Receptor-neonate (FcRn)binding domain (FcRn^(HIGH) mutations). The FcRn binding domain has beenimplicated in the maternal-fetal transfer of antibodies. See Story etal., J. Exp. Med., 180:2377 2381, 1994. The amino acid substitutions inthe FcRn binding domain increase the affinity of the constant domain forthe FcRn thereby increasing the half-life of the antibody.

FcRn binding domain mutations M252Y, S254T and T256E (YTE) have beendescribed to increase FcRn binding and thus increase the half-life ofantibodies. See U.S. Published Patent Application No. 2003/0190311 andDall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006). Additionally,FcRn binding domain mutations N434A and Y436I (AI) have been describedto also increase FcRn binding. See Yeung et al., J. Immunol. 182:7663-7671 (2009). The YTE (M252Y/S254T/T256E) and AI (N434/Y436I)mutations were incorporated into both anti-TfR^(52A)/BACE1 andanti-TfR²/BACE1 bispecific antibodies containing either WT human IgG1 oreffectorless LALAPG or N297G mutations. In addition, FcRn^(HIGH)mutation were made in the anti-gD hIgG1 antibody as a control. Mutationswere constructed using Kunkel mutatgenesis, antibodies were expressedtransiently in CHO cells, and proteins were purified using protein Achromatography followed by size exclusion chromatography (SEC).

Binding of FcRn^(HIGH) variant antibodies to FcRn was measured usingBlAcore. Human and cynomolgus monkey FcRn proteins were expressed in CHOand purified using IgG affinity chromatography. Data were acquired on aBlAcore T200 instrument. A series S sensor chip CM5 (GE Healthcare, Cat.BR100530) was activated with EDC and NHS reagents according to thesupplier's instructions, and anti-Fab antibody (Human Fab capture kit,GE Health care Bio-science. AB SE-75184, upsala, Sweden) was coupled toachieve approximately 10,000 response units (RU), followed by blockingun-reacted groups with 1 Methanolamine. For affinity measurements,antibodies were first injected at a 10 μl/min flow rate to captureapproximately 1000 RU on 3 different flow cells (FC), except for FC1(reference), and then 2-fold serial dilutions of human FcRn (or CynoFcRn) in pH6 buffer (0.1M sodium phosphate), from low (1 nM) to high (25μM) were injected (flow rate: 30 μl/min) one after the other in the samecycle with no regeneration between injections. Sensograms were recordedand subject to reference and buffer subtraction before evaluating byusing BlAcore T200 Evaluation Software (version 2.0). Affinities weredetermined by analyzing the level of binding at steady state based on a1:1 binding model. Binding affinities for LALAPG, N297G, LALAPG.YTE, andLALAPG.AI variants of anti-TfR^(52A)/BACE1 are shown in Table 11 below.The data show that the FcRn^(HIGH) variants enhance affinity atendosomal (pH 6) to both human and cyno FcRn.

TABLE 11 Human Cyno FcRn FcRn Effector KD at pH 6 KD at pH 6 AntibodyFuncion FcRn High (uM) (uM) Anti-TfR.52A/BACE1.hIgG1 WT WTAnti-TfR.52A/BACE1.hIgG1.N297G N297G WT 1.3 2.1Anti-TfR.52A/BACE1.hIgG1.LALAPG LALAPG WT 0.8 1.2Anti-TfR.52A/BACE1.hIgG1.N297G.YTE N297G YTEAnti-TfR.52A/BACE1.hIgG1.LALAPG.YTE LALAPG YTE 0.2 0.2Anti-TfR.52A/BACE1.hIgG1.N297G.AI N297G N434A/Y436IAnti-TfR.52A/BACE1.hIgLALAPG.AI LALAPG N434A/Y436I 0.6 0.4Anti-TfR.52A/BACE1.hIgG1.N297G.A N297G N434A Anti-TfR2/BACE1.hIgG1.N297GN297G WT 1.7 2.1 Anti-TfR2/BACE1.hIgG1.LALAPG LALAPG WT 1.1 1.2Anti-TfR2/BACE1.hIgG1.LALAPG.YTE LALAPG YTE 0.3 0.2 Anti-gD.hIgG1 WT WT0.7 0.9 Anti-gD.hIgG1.YTE WT YTE Anti-gD.hIgG1.AI WT N434A/Y436I 0.3 0.4Anti-gD.hIgG1.A WT N434A 0.1 0.7

Select FcRnHIGH variants will be tested in cynomologus monkeys todetermine whether enhancement of FcRn affinity can increase improve thepharmacokinetic properties and/or increase the brain exposure of theanti-TfR/BACE1 antibodies.

To evaluate the safety of the effectorless and FcRn^(HIGH) mutations,certain bispecific antibodies were administered to human transferrinreceptor knock-in mice which express the human transferrin receptor. ThehuTfR knock-in mice were generated as follows. The construct fortargeting human TFRC cDNA into the C57BL/6 Tfrc locus in ES cells wasmade using a combination of recombineering (Warming et al. Molecular andCellular Biology vol. 26 (18) pp. 6913-22 2006; Liu et al GenomeResearch (2003) vol. 13 (3) pp. 476-84) and standard molecular cloningtechniques.

Briefly, a cassette (human TFRC cDNA, SV40 pA, andfrt-PGK-em7-Neo-BGHpA-frt) flanked by short homologies to the mouse Tfrcgene was used to modify a Tfrc C57BL/6J BAC (RP23 BAC library) byrecombineering. The human TFRC cDNA cassette was inserted at theendogenous ATG and the remainder of Tfrc exon 2 plus the beginning ofintron 2 was deleted. The targeted region in the BAC was then retrievedinto pBlight-TK (Warming et al. Molecular and Cellular Biology vol. 26(18) pp. 6913-22 2006) along with flanking genomic Tfrc sequences ashomology arms for ES cell targeting. Specifically, the 2950 bp 5′homology arm corresponds to (assembly NCBI37/mm9):chr.16:32,610,333-32,613,282 and the 2599 bp 3′ homology arm correspondsto chr.16:32,613,320-32,615,918. The final vector was confirmed by DNAsequencing.

The Tfrc/TFRC KI vector was linearized with NotI and C57BL/6N C2 EScells were targeted using standard methods (G418 positive andgancyclovir negative selection). Positive clones were identified usingPCR and taqman analysis, and confirmed by sequencing of the modifiedlocus. Correctly targeted ES cells were transfected with a Flpe plasmidto remove Neo and ES cells were then injected into blastocysts usingstandard techniques. Germline transmission was obtained after crossingresulting chimaeras with C57BL/6N females.

Specifically, the antibodies listed in the table below were administeredto huTfR knock-in mice in a single 50 mg/kg dose and 24 hours laterblood was drawn and reticulocytes. huIg1, N297G

TABLE 12 Antibody Isotype Number of Mice Anti-gD huIg1, N297G 6anti-TfR^(52A)/BACE1 huIgG1, N297G 6 anti-TfR^(52A)/BACE1 huIgG1, LALAPG6 anti-TfR^(52A)/BACE1 huIgG1, LALAPG/YTE 6 anti-TfR^(52A)/BACE1 huIgG1,LALAPG/AI 6

Following administration of the anti-TfR^(52A)/BACE1 LALAPG, LALAPG/YTEor LALAPG/AI antibodies (Groups 3-5 in Table 12), human TfR knock-inmice displayed no clinical symptoms or reticulocyte loss (FIG. 22) aspreviously observed using anti-TfR antibodies with full effectorfunction (Couch et al. 2013). These results indicate that incorporationof the LALAPG mutation on the human IgG1 framework also abrogateseffector function, and further suggest that addition of either the YTEor AI FcRn^(HIGH) mutations do not interfere with the desired propertiesof the LALAPG mutations to render the antibody effectorless.

ADCC assays were also carried out to confirm the effectorless status ofLALAPG, LALAPG/YTE, and LALAPG/AI mutation combinations in ahuman-derived cell line. As previously, human erythroleukemia cell line(HEL, ATCC) was used as target cells with PBMCs from healthy humandonors carrying either the F/V158 genotype or the FcγRIIIA V/V158genotype. The V/V158 genotype was also included in this assay due to theknown association with increased NK cell-mediated ADCC activity as wellas ability to bind IgG4 antibodies (Bowles and Weiner, 2005; Bruhns etal. 2008). Cells were counted and viability was determined by Vi-CELL®(Beckman Coulter; Fullerton, Calif.) following the manufacturer'sinstructions.

PBMCs were isolated by density gradient centrifugation using Uni-Sep™blood separation tubes (Accurate Chemical & Scientific Corp.; Westbury,N.Y.). Target cells in 50 μL of assay medium (RPMI-1640 with 1% BSA and100 units/mL penicillin and streptomycin) were seeded in a 96-well,round-bottom plate at 4×10⁴/well. Serial dilutions of test and controlantibodies (50 μL/well) were added to the plates containing the targetcells, followed by incubation at 37° C. with 5% CO₂ for 30 minutes toallow opsonization. The final concentrations of antibodies ranged from0.0051 to 10,000 ng/mL following 5-fold serial dilutions for a total of10 data points. After the incubation, 1.0×10⁶ PBMC effector cells in 100μL of assay medium were added to each well to give a ratio of 25:1effector: target cells, and the plates were incubated for an additional4 hours. The plates were centrifuged at the end of incubation and thesupernatants were tested for lactate dehydrogenase (LDH) activity usinga Cytotoxicity Detection Kit™ (Roche Applied Science; Indianapolis,Ind.). The LDH reaction mixture was added to the supernatants and theplates were incubated at room temperature for 15 minutes with constantshaking. The reaction was terminated with 1 M H₃PO₄, and absorbance wasmeasured at 490 nm (the background, measured at 650 nm was subtractedfor each well) using a SpectraMax Plus microplate reader. Absorbance ofwells containing only the target cells served as the control for thebackground (low control), whereas wells containing target cells lysedwith Triton-X100 provided the maximum signal available (high control).Antibody-independent cellular cytotoxicity (AICC) was measured in wellscontaining target and effector cells without the addition of antibody.The extent of specific ADCC was calculated as follows:

${\%\mspace{14mu} A\; D\; C\; C} = {100 \times \frac{{A_{490}\mspace{14mu}({Sample})} - {A_{490}\mspace{11mu}\left( {A\; I\; C\; C} \right)}}{{A_{490}\mspace{14mu}\left( {{High}\mspace{14mu}{Control}} \right)} - {A_{490}\mspace{14mu}\left( {{Low}\mspace{14mu}{Control}} \right)}}}$ADCC values of sample dilutions were plotted against the antibodyconcentration, and the dose-response curves were fitted to afour-parameter model using SoftMax Pro.

Results of the ADCC assay are shown in FIG. 23. As expected, theeffector positive anti-human TfR antibody (anti-TfR¹/gD IgG1 WT)elicited significant ADCC activity on the HEL cells. In contrast, theanti-TfR^(52A)/BACE1 antibody variants containing LALAPG, LALAPG/YTE, orLALAPG/AI mutations did not display any ADCC activity in HEL cells,similar to the negative control anti-TfR^(52A)/gD N297G antibody.

Example 9: Modification of Anti-TfR Bispecific Antibody Sequences

A mutation in heavy chain variable region FR4, methionine at position108 changed to leucine (M108L), may be incorporated to promote stabilityof an anti-TfR antibody. Binding affinity of an anti-TfR Fab comprisingthe M108L mutation was similar to the unmodified Fab, as shown in Table13.

TABLE 13 Binding affinity of anti-TfR Fab ka (1/Ms) kd (1/s) KD (M)antiTfR.52A.Fab 3.71E+05 0.0204 5.50E−08 antiTfR.52A.M108L.Fab 3.20E+050.02312 7.22E−08

It is expected that a bispecific antibody comprising an anti-TfR armcomprising an anti-TfR².hIgG1.LALAPG.YTE heavy chain having an M108Lmutation (SEQ ID NO: 160) will be more stable than the bispecificantibody comprising the same anti-TfR arm without the M108L heavy chainmutation, while also retaining binding affinity for TfR.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Certain Sequences SEQ ID NO Descripton Sequence 158Anti-TfR^(52A) M108L heavy EVQLVQSGAE VKKPGASVKV SCKASGYTFTchain variable region SYWMHWVRQA PGQRLEWIGE IAPTNGRTNYIEKFKSRATL TVDKSASTAY MELSSLRSED TAVYYCARGT RAYHYWGQGT LVTVSS 159Anti-TfR² M108L heavy chain EVQLVQSGAE VKKPGASVKV SCKASGYTFTvariable region SYWMHWVRQA PGQRLEWIGE INPTNGRTNYIEKFKSRATL TVDKSASTAY MELSSLRSED TAVYYCARGT RAYHYWGQGT LVTVSS 160 Anti-EVQLVQSGAE VKKPGASVKV SCKASGYTFT TfR²/Ag2.hIgG1.LALAPG.YTE.SYWMHWVRQA PGQRLEWIGE INPTNGRTNY M108L bispecific antibodyIEKFKSRATL TVDKSASTAY MELSSLRSED anti-TfR heavy chainTAVYYCARGT RAYHYWGQGT LVTVSSASTK Ag2 = antigen 2GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYSLSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PEAAGGPSVFLFPPKPKDTL YITREPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALGAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLSCAVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLVSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK 161 Anti- DIQMTQSPSS LSASVGDRVT ITCRASDNLYTfR²/Ag2.hIgG1.LALAPG.YTE SNLAWYQQKP GKSPKLLVYD ATNLADGVPSbispecific antibody anti- RFSGSGSGTD YTLTISSLQP EDFATYYCQHTfR light chain FAGTPLTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 162 Anti-TfR^(52A) light chainDIQMTQSPSS LSASVGDRVT ITCRASDNLY variable regionSNLAWYQQKP GKSPKLLVYD ATNLADGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQHFWGTPLTFGQ GTKVEIK 163 Anti-TfR² light chainDIQMTQSPSS LSASVGDRVT ITCRASDNLY variable regionSNLAWYQQKP GKSPKLLVYD ATNLADGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQHFAGTPLTFGQ GTKVEIK

What is claimed is:
 1. An isolated antibody that binds to humantransferrin receptor (TfR) and primate TfR, wherein the antibodycomprises a heavy chain variable region amino acid sequence of SEQ IDNO: 158 or SEQ ID NO: 159, and a light chain variable region amino acidsequence of SEQ ID NO: 162 or SEQ ID NO:
 163. 2. The isolated antibodyof claim 1, wherein the antibody comprises a heavy chain variable regionamino acid sequence of SEQ ID NO: 158 and a light chain variable regionamino acid sequence of SEQ ID NO: 162, or a heavy chain variable regionamino acid sequence of SEQ ID NO: 159 and a light chain variable regionamino acid sequence of SEQ ID NO:
 163. 3. The antibody of claim 1, whichis a monoclonal antibody.
 4. The antibody of claim 1, wherein theantibody comprises at least one mutation in the antibody Fc region,relative to a wild-type antibody Fc region of the same isotype, thatreduces or eliminates the complement activation function of the antibodyor the effector function of the antibody, or increases the half-life ofthe antibody.
 5. The antibody of claim 4, wherein the at least onemutation reduces or eliminates the effector function or complementactivation function relative to an antibody comprising a wild-typeantibody Fc region of the same isotype.
 6. The antibody of claim 4,wherein the at least one mutation reduces or eliminates the effectorfunction relative to an antibody comprising a wild-type antibody Fcregion of the same isotype.
 7. The antibody of claim 4, wherein the atleast one mutation increases the half-life of the antibody.
 8. Theantibody of claim 7, wherein the half-life is increased by at least onemutation in the FcRn binding domain of the antibody at a positionselected from: 252, 254, 256, 434 and 436, wherein amino acid numberingis according to the EU numbering system.
 9. The antibody of claim 8,wherein the FcRn binding domain comprises mutations at positions 252,254 and
 256. 10. The antibody of claim 9, wherein the FcRn bindingdomain comprises M252Y, S254T and T256E mutations.
 11. The antibody ofclaim 8, wherein the FcRn binding domain comprise mutations at positions434 and
 436. 12. The antibody of claim 11, wherein the FcRn bindingdomains comprises N434A and Y436I mutations.
 13. The antibody of claim1, wherein the antibody is of an isotype that naturally has reduced oreliminated effector function.
 14. The antibody of claim 1, wherein theglycosylation of the antibody is reduced by a method selected from:production of the antibody in an environment that does not permitwild-type glycosylation; removal of carbohydrate groups already presenton the antibody; and mutation of a glycosylation site of the antibodysuch that wild-type glycosylation does not occur.
 15. The antibody ofclaim 14, wherein the antibody is produced in a non-mammalian cellproduction system, or where the antibody is produced synthetically. 16.The antibody of claim 14, wherein the Fc region of the antibodycomprises a mutation at position 297 such that the wild-type asparagineresidue at that position is replaced with another amino acid thatinterferes with glycosylation at that position, wherein amino acidnumbering is according to the EU numbering system.
 17. The antibody ofclaim 1, wherein the effector function or complement activation functionis reduced or eliminated by deletion of all or a portion of the Fcregion.
 18. The antibody of claim 1, wherein the antibody comprises atleast one mutation is-selected from: a point mutation of the Fc regionto impair binding to one or more Fc receptors selected from thefollowing positions: 234, 235, 238, 239, 248, 249, 252, 254, 265, 268,269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303,322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414,416, 419, 434, 435, 437, 438, and 439; a point mutation of the Fc regionto impair binding to C1q selected from the following positions: 270,322, 329, and 321; and a point mutation at position 132 of the CH1domain, wherein amino acid numbering is according to the EU numberingsystem.
 19. The antibody of claim 18, wherein the antibody comprises atleast one point mutation of the Fc region to impair binding to one ormore Fc receptors selected from 234, 235, 265, 297 and
 329. 20. Theantibody of claim 19, wherein the Fc region comprises a mutation atposition 297 or at positions 265 and
 297. 21. The antibody of claim 19,wherein the Fc region comprises mutations at positions 234, 235 and 329.22. The antibody of claim 19, wherein the Fc region comprises a N297Gmutation; D265A and N297A mutations; or D265A and N297G mutations. 23.The antibody of claim 19, wherein the Fc region comprises L234A, L235A,and P329G mutations.
 24. The antibody of claim 1, wherein the antibodyhas a KD for TfR of about 20 nM to 100 nM.
 25. The antibody of claim 1,wherein the antibody is coupled to a therapeutic compound.
 26. Theantibody of claim 25, wherein the antibody is a multispecific antibodyand the therapeutic compound optionally forms one portion of themultispecific antibody.
 27. The antibody of claim 26, wherein themultispecific antibody comprises a first antigen binding site whichbinds TfR and a second antigen binding site which binds a brain antigen.28. The antibody of claim 27, wherein the multispecific antibodycomprises a first heavy chain comprising the sequence of SEQ ID NO: 160and a first light chain comprising the sequence of SEQ ID NO:
 161. 29.The antibody of claim 27, wherein the brain antigen is selected from thegroup consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growthfactor receptor (EGFR), human epidermal growth factor receptor 2 (HER2),tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6.
 30. The antibody of claim 27, wherein the multispecific antibodybinds both TfR and BACE1.
 31. The antibody of claim 27, wherein themultispecific antibody binds both TfR and Abeta.
 32. The antibody ofclaim 25, wherein the therapeutic compound is a neurological disorderdrug.
 33. A method of transporting a compound across the blood-brainbarrier (BBB) in a subject comprising exposing the BBB to an antibody ofclaim 25 such that the antibody transports the compound coupled theretoacross the BBB.
 34. A method of increasing exposure of the centralnervous system CNS of a subject to a compound, comprising exposing theCNS to an antibody of claim 25 such that the antibody transports thecompound coupled thereto to the CNS.
 35. A method of increasingretention in the CNS of a compound administered to a subject, comprisingexposing the CNS to an antibody of claim 25 such that the retention inthe CNS of the compound is increased.
 36. A pharmaceutical formulationcomprising the antibody of claim 1 and a pharmaceutically acceptablecarrier.
 37. An isolated nucleic acid encoding the antibody of claim 1.38. A host cell comprising the nucleic acid of claim
 37. 39. A method ofproducing an antibody comprising culturing the host cell of claim 38 sothat the antibody is produced and optionally further comprisingrecovering the antibody from the host cell.
 40. An isolated antibodyfragment that binds to human transferrin receptor (TfR) and primate TfR,wherein the antibody fragment comprises a heavy chain variable regionamino acid sequence of SEQ ID NO: 158 or SEQ ID NO: 159, and a lightchain variable region amino acid sequence of SEQ ID NO: 162 or SEQ IDNO: 163.